Lactobacillus acidophilus surface layer protein a (SlpA) as a therapeutic agent for the treatment of inflammatory diseases

ABSTRACT

The current invention provides a recombinant bacterium, the recombinant bacterium being genetically modified to decrease or eliminate the display of lipoteichoic acid (LTA), surface layer protein B (SlpB) and surface layer protein X (SlpX) on the surface of said bacterium. Efficacious therapies for a subject suffering from an inflammation mediated disease are also provided. The methods of the current invention comprise administering to a subject in need thereof a therapeutically effective amount of the recombinant  L. acidophilus  cells or a therapeutically effective amount of the isolated surface layer protein A (SlpA) or a non-naturally occurring derivative thereof. The recombinant  L. acidophilus  cells or SlpA isolated from  L. acidophilus  can be in a pharmaceutical composition comprising a pharmaceutically acceptable carrier and/or excipient. In an embodiment of the invention, the pharmaceutical composition is administered orally.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/790,793, filed Feb. 14, 2020, which is a divisional of U.S.application Ser. No. 15/521,418, filed Apr. 24, 2017, now U.S. Pat. No.10,562,943, which is the U.S. national stage application ofInternational Patent Application No. PCT/US2015/055620, filed Oct. 15,2015, which claims the benefit of U.S. Provisional Application Ser. No.62/068,338, filed Oct. 24, 2014, the disclosures of which are herebyincorporated by reference in their entirety, including all figures,tables and amino acid or nucleic acid sequences.

The Sequence Listing for this application is labeled “Seq-Liste.txt”which was created on Jan. 12, 2019 and is 63 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

GOVERNMENT SUPPORT STATEMENT

This invention was made with government support under A1093370 awardedby The National Institutes of Health and under W81XWH-12-1-0368 awardedby the United States Department of Defense. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

The gastrointestinal (GI) microbiota plays a critical role indetermining the immunologic outcome of various signaling events in hostcells via their gene products, exceeding the human genome by ahundredfold (Ley et al., 2006; Qin et al., 2010). As such, thecomposition of the GI microbiota and host immunity are mutualistic andcontinuously influence each other (Maslowski and Mackay, 2011; McDermottand Huffnagle, 2014).

Intestinal homeostasis is tightly controlled by regulatory immunemechanisms, which are established by the interactions of the trillionsof microbes and their gene products with numerous pattern recognitionreceptors (PRRs), including C-type lectin receptors (CLRs), such asSIGNR3 (Konstantinov et al., 2008; Osorio and Reis e Sousa, 2011).Disruption of this delicate balance by inimical signals has devastatingconsequences that may result in intestinal disorders, includinginflammatory bowel disease (IBD). When this occurs, highly activatedinnate cells trigger intestine-infiltrating pathogenic T cell subsets(e.g., Th1, Th17), and regulatory T cells (Tregs) with pro-inflammatorycharacteristics (Geremia et al., 2014; Khazaie et al., 2012; Neurath,2014) that ultimately drive tissue destruction and intestinal diseaseprogression. Innate cells (e.g., dendritic cells, macrophages) are theinitial targets of either culpable microbes or their gene products,which subsequently affect the regulation/stimulation of intestinalimmunity (Atarashi et al., 2013; Ivanov and Honda, 2012; Yang et al.,2014). Given these entwined relationships, it is not surprising thatmicrobial products have been linked to the pathology of intestinalauto-inflammation (Nicholson et al., 2012). The underlying associationsbetween gut microbes and inflammatory diseases (e.g., IBD) have beenwell documented; however, the cellular and molecular mechanisms by whichintestinal commensal gene product(s) and their molecular receptor(s)impact immune responses remain unclear.

Information regarding the immunobiologic functions of Lactobacillusacidophilus surface layer proteins (Slps) is relatively limited. Slpsare paracrystalline (glyco) protein arrays that are abundantly presenton the cell surfaces of most eubacteria and archaea, including L.acidophilus (Johnson et al., 2013). L. acidophilus NCFM possesses threeSlp-encoding genes: slpA (LBA0169), slpB (LBA0175), and slpX (LBA0512)(Goh et al., 2009). Diverse functional roles have been proposed forSlps, including cell shape determinants, molecular sieves, protectivelayers against viral infection, anchoring sites for surface-associatedenzymes and facilitators of cellular adhesion through PRRs, includingC-type lectins (CLECs) (Konstantinov et al., 2008).

CLECs recognize carbohydrate structures on self and non-self antigens(Engering et al., 2002; Osorio and Reis e Sousa, 2011). Eighteen CLECs,including DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), have beenidentified on dendritic cells (DCs) and macrophages (MΦs) (Ehlers, 2010;van Kooyk and Geijtenbeek, 2003). DC-SIGN, which was previously shown tobind L. acidophilus-SlpA in vitro (Konstantinov et al., 2008), is acalcium-dependent carbohydrate-binding protein with specificity for themannose-containing glycans of microbial surface components andfucose-containing Lewis antigens (Ehlers, 2010). Of the eight murinehomologs of DC-SIGN, SIGNR3 (CD209d) exhibits the most biochemicalsimilarity to human DC-SIGN (Powlesland et al., 2006).

SIGNR3 contains a carbohydrate recognition domain (CRD) and signalsthrough a hemi-immunoreceptor tyrosine-based activation motif(hemi-ITAM) pathway (Tanne et al., 2009). Such signaling potentiallydownregulates the ubiquitously expressed leukotriene A₄ hydrolase(LTA₄H) (Tobin et al., 2010) that catalyzes proinflammatory leukotrieneB₄ (LTB₄) synthesis from LTA₄ (Snelgrove et al., 2010), whichconsequently activates interleukin (IL)-1β production. Here, we identifySlpA as a key effector molecule expressed by L. acidophilus, anddemonstrate its in vivo protective role in murine colitis models.Moreover, we provide evidence that protection by L. acidophilus-SlpA isconferred via signaling through a single CLR, namely SIGNR3.

As discussed above, normal gut immune responses dictate that residentinnate and adaptive immune cells must coexist with the large number ofmicrobes inhabiting the GI tract while still being able to mount animmune response against invading pathogens. Maintenance of immunehomeostasis toward commensal bacteria and their microbial gene productsis essential in the prevention of chronic inflammation in the gut. Overtintestinal inflammation is a hallmark of IBD. Current therapies for themanagement of IBD include antibiotic regimens to prevent the outgrowthand systemic dissemination of pathogenic microorganisms, as well ascorticosteroids and immunomodulators to decrease the inflammatoryresponse in the intestines. However, these therapies are not withoutundesirable and harmful side effects, as antibiotics also deplete thebeneficial intestinal microflora, and corticosteroids andimmunomodulators act as global immune suppressors, thereby increasingthe risk of infection and cancer. Thus, there is a need for identifyingnew therapeutic agents for the treatment of such diseases.

BRIEF SUMMARY OF THE INVENTION

The current invention provides a recombinant bacterium, for example, arecombinant Lactobacillus acidophilus, the recombinant bacterium beinggenetically modified to decrease or eliminate the display oflipoteichoic acid (LTA), surface layer protein B (SlpB) and surfacelayer protein X (SlpX) on the surface of said bacterium.

The current invention also provides an efficacious therapy for a subjectsuffering from an inflammation mediated disease (inflammatory diseases),for example, an autoinflammatory disease, such as IBD or otherinflammatory diseases, such as allergies, ankylosing spondylitis,Crohn's disease, diabetes, Type I diabetes, gastroesophageal refluxdisease, Hashimoto's thyroiditis, hyperthyroidism, hypothyroidism,interstitial cystitis (IC), Löfgren's syndrome, lupus erythematosis,myasthenia gravis, multiple sclerosis, osteoarthritis, polymyalgiarheumatica, prostatitis, psoriasis, psoriatic arthritis, Raynaud'ssyndrome/phenomenon, reactive arthritis (Reiter syndrome), restless legsyndrome, reflex sympathetic dystrophy (RSD), rheumatoid arthritis,scleroderma, Sjögren's syndrome, ulcerative colitis and uveitis. Themethods of the current invention comprise administering to a subject inneed thereof a therapeutically effective amount of recombinant L.acidophilus cells of the current invention or a therapeuticallyeffective amount of purified surface layer protein A (SlpA), forexample, SlpA isolated from L. acidophilus. In one embodiment, the L.acidophilus cells belong to L. acidophilus strain NCK2187 which is abacterium genetically modified to decrease or eliminate the display ofLTA, SlpB and SlpX on the surface. Another embodiment provides for therecombinant expression of SlpA in bacterial cells that are devoid ofLTA, SlpB and SlpX expression on the cell surface.

The recombinant bacterial cells (e.g., L. acidophilus cells) or SlpAisolated from L. acidophilus can be formulated into a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and/orexcipient (optionally in combination with other therapeutic agents). Inan embodiment of the invention, the pharmaceutical composition isadministered orally.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication, withcolor drawing(s), will be provided by the Office upon request andpayment of the necessary fee.

FIGS. 1A-1E. L. acidophilus NCK2187 strain development andcharacteristics. A. Agarose gel image illustrating PCR amplicons of ltaS(LBA0447), slpB, and slpX deletions in NCK2187. B. SDS-PAGE gel of 5 MLiCl-purified S-layer fractions from the parental strains, NCK56 andNCK1909; NCK2030 (LTA⁺, SlpA⁺, SlpB⁻, SlpX⁻); and NCK2187 (LTA⁻, SlpA⁺,SlpB⁻, SlpX⁻). C. Protein gel showing predominance of SlpA in NCK2187and absence of other Slps. D. B6 mice were orally gavaged with 10⁹ CFUerythromycin-resistant NCK56 or NCK2187. Fecal pellets were collecteddaily and tested for the presence of erythromycin-resistant strains. n=3mice/group. Data are representative of five independent experiments andare shown as mean±SEM. E. Colonic LP cells were co-cultured with NCK56or NCK2187 (1:1) and secreted cytokines were measured in thesupernatants. Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001.

FIGS. 2A-2C. L. acidophilus NCK2187 promotes intestinal regulation insteady-state. A. B6 mice were orally gavaged with 10⁹ CFU NCK56 (blue)or NCK2187 (green) on days 0, 3, 6, and 9, or left untreated, and immuneresponses in the colon analyzed at day 14 by flow cytometry. B-C.FoxP3-GFP mice were treated and evaluated as in (A). C. Regulatorycytokine production in FoxP3-GFP⁺ (green dotted bars) versus FoxP3-GFP⁻(white bars) cells was measured by intracellular staining and FACSanalyses. n=5 mice/group. Data represent four individual experiments andare shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.Black asterisks compare NCK2187 to untreated (PBS) mice, and redasterisks to NCK56-treated mice.

FIGS. 3A-3D. L. acidophilus NCK2187 and its SlpA protect againstpathogenic T-cell induced colitis. Rag1^(−/−) mice were injected with10⁶ CD4⁺CD45RB^(hi) T cells, and then orally gavaged with NCK56 (red),NCK2187 (green), or SlpA (blue), 1 and 3 days after transfer, andsubsequently once a week for 4 consecutive weeks, or left untreated(magenta). A group of mice was co-transferred with CD4⁺CD25⁺ T cells asa positive control for protection (Tregs; gray). Colitis severity wasdetermined in part by weight loss, diarrhea scores, and FOB (A). (SeeTables 2-4 for statistical analyses results.) B-C. Colitis scores basedon histopathology and gross morphology of the colons were also used asmeasures of disease. Scale bar=200 μm. D. Circulating levels ofproinflammatory cytokines were measured in the sera of the micetransferred and treated as mentioned above, or sham adoptive transferred(white bars). n=5 mice/group. Data represent three individualexperiments and are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001,****P<0.0001. Black asterisks compare NCK2187 to PB S-treated adoptivelytransferred mice, and red asterisks to NCK56-treated mice.

FIGS. 4A-4D. L. acidophilus NCK2187 and its SlpA protect intestinalbarrier function and prevent dysbiosis in pathogenic T-cell inducedcolitis. Rag1^(−/−) mice were injected with 10⁶ CD4⁺CD45RB^(hi) T cells,and then orally gavaged with NCK56 (red), NCK2187 (green), or SlpA(blue), 1 and 3 days after transfer, and subsequently once a week for 4consecutive weeks, or left untreated (magenta). A group of mice wasco-transferred with CD4⁺CD25⁺ T cells as a positive control forprotection (Tregs; gray). A. Colonic expression of tightjunction-associated genes Cldn3 and Ocln, determined by RT-PCR, as wellas passive transepithelial absorption of FITC-dextran, were used asmeasures of epithelial barrier integrity. Sham adoptive transferredRag1^(−/−) mice (white bars) were used as baseline controls in somecases. n=5 mice/group. Data represent three individual experiments andare shown as mean±SEM. *P<0.05, **P<0.01. Black asterisks compareNCK2187 to PBS-treated adoptively transferred mice, and red asterisks toNCK56-treated mice. B. UniFrac analyses were used to calculate distancesbetween the microbial communities of the different samples (week 7) andthree-dimensional scatterplots were generated by using principalcoordinate analyses (PCoA). Gray dots=CD4⁺CD45RB^(hi) T cells+PBS;yellow dots=CD4⁺CD45RB^(hi) T cells+NCK56; green dots=CD4⁺CD45RB^(hi) Tcells+NCK2187; blue dots=CD4⁺CD45RB^(h1) T cells+SlpA; reddots=CD4⁺CD45RB^(hi) T cells+Tregs. Each dot represents the fecalmicrobiota data of an individual mouse. C. Changes in the relativeabundance of different phyla were also analyzed. (See Table 5 forstatistical analyses among the different groups.) Darkblue=Actinobacteria; green=Bacteroidetes; red=Proteobacteria;yellow=Verrucomicrobia; aqua=Firmicutes; purple=Tenericutes;black=Others. D. Comparison of microbial communities at family or orderlevels. The heat map depicts the relative value in individual mice.n=5-6 mice/group.

FIGS. 5A-5D. L. acidophilus NCK2187 and its SlpA bind to murine SIGNR3to induce regulatory signals. A. B6 mice were orally gavaged with 10⁹CFU NCK56 or NCK2187 and the colonic gene expression of C-type lectinreceptors were measured by RT-PCR. Each box represents an individualmouse; n=4. Data represent three individual experiments and are shown asmean±SEM. B. Binding of SlpA to various hFc fusion proteins wereanalyzed by flow cytometry. Gray tinted line=SlpA-coated beads only;orange=SlpA-coated beads+secondary antibody; green=SlpA-coatedbeads+control fusion protein; blue=SlpA-coated beads+SIGNR1-hFc;red=SlpA-coated beads+SIGNR3-hFc. Binding assays results were confirmedfive independent times. C. IL-1β production by colonic DCs of naïve WTB6 GF or Signr3^(−/−) mice treated with NCK56 (blue) or NCK2187 (green)on days 0, 3, 6, and 9, or left untreated (black), as determined by flowcytometry. D. Frequency of colonic FoxP3⁺ Tregs in KO mice treated withNCK56 or NCK2187 was measured by flow cytometry. n=5 mice/group. Datarepresent four individual experiments and are shown as mean±SEM.*P<0.05. Black asterisks compare NCK2187 to untreated (PBS) mice, andred asterisks to NCK56-treated mice.

FIGS. 6A-61I. L. acidophilus NCK2187 and its SlpA do not protect againstDSS-induced colitis in Signr3^(−/−) mice. WT or Signr3^(−/−) (KO) micewere orally gavaged with NCK56, NCK2187 or SlpA at days −1 and −3, and3% DSS was given in the drinking water. Mice were gavaged with bacteriaor purified SlpA an additional 2 times and monitored for diseaseprogression. Colitis severity was determined in part by weight loss (A).(See Table 6 for statistical analyses from WT mice.) B-C. Colitis scoresbased on histopathology, and gross morphology of the colons were alsoused as measures of disease. Scale bar=200 μm. n=5 mice/group. Emptybars=WT; lined bars=KO; white bars=untreated; purple bars=DSS; redbars=DSS+NCK56; green bars=DSS+NCK2187; blue bars=DSS+SlpA. D.Colonoscopies were performed in the different groups with aMulti-Purpose Rigid™ Telescope attached to a TELE PACK X. E. Meanrelative colonic expression of tight junction-associated genes in WTmice. F. Fecal albumin levels in WT mice as a measure of intestinalpermeability. G. UniFrac analyses were used to calculate distancesbetween the microbial communities of the different samples (Day 10) andthree-dimensional scatterplots were generated by using PCoA. Light gray:WT+DSS; green: WT+DSS+NCK56; brown: WT+DSS+NCK2187; blue: WT+DSS+SlpA;aqua: KO+DSS; red: KO+DSS+NCK56; yellow: KO+DSS+NCK2187; purple:KO+DSS+SlpA. n=4-6 mice/group. H. Species richness and microbialdiversity in DSS-treated mice. Top: The Chao richness index was used asa measure of species richness. Bottom: The Shannon diversity index wasused to estimate microbial diversity for each group. Data are shown asmean±SEM. *P<0.05, **P<0.01, ****P<0.0001.

FIGS. 7A-7C. L. acidophilus NCK2187 and its SlpA do not prevent immuneinfiltration and activation during DSS-induced colitis in Signr3^(−/−)mice. WT or Signr3^(−/−) (KO) mice were orally gavaged with NCK56,NCK2187 or SlpA at days −1 and −3, and 3% DSS was given in the drinkingwater. Mice were gavaged with bacteria or purified SlpA an additional 2times, and immunity analyzed by flow cytometry at day 10. A.Representative plots indicate the frequency of neutrophils in the colonsof untreated or DSS-treated WT (left) and Signr3^(−/−) mice (right).B-C. Colonic DCs and MΦs were analyzed by flow cytometry for theproduction of IL-1β (B), and colonic Tregs were evaluated forco-expression of RORγt⁺ (C). n=5 mice/group. Gray tinted line=isotypecontrol; black=untreated; purple=DSS; red=DSS+NCK56; green=DSS+NCK2187;blue=DSS+SlpA. Data are shown as mean±SEM, and are representative ofthree different experiments. *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.

FIGS. 8A-8B. L. acidophilus NCK2187 and its SlpA in pathogenic T-cellinduced colitis. Rag1^(−/−) mice were injected with 10⁶ CD4+CD45RBhi Tcells, and then orally gavaged with NCK56 (red), NCK2187 (green), orSlpA (blue), 1 and 3 days after transfer, and subsequently once a weekfor 4 consecutive weeks, or left untreated (magenta). A group of micewas co-transferred with CD4⁺CD25⁺ T cells as a positive control forprotection (Tregs; gray). A. Colonic expression of Ltb4r1, Ltb4r2, andIl1b were determined by RT-PCR. n=5 mice/group. Data represent threeindividual experiments and are shown as mean±SEM. *P<0.05, **P<0.01.Black asterisks compare NCK2187 to PBS-treated adoptively transferredmice, and red asterisks to NCK56-treated mice. B. Colonoscopies wereperformed in the specified groups with a Multi-Purpose Rigid™ Telescopeattached to a TELEPACK X.

FIG. 9. L. acidophilus NCK2187 and its SlpA cannot normalize seracytokine levels upon DSS-induced colitis in Signr3^(−/−) mice. WT orSignr3^(−/−) mice were orally gavaged with NCK56, NCK2187 or SlpA atdays −1 and −3, and 3% DSS was given in the drinking water. Mice weregavaged with bacteria or purified SlpA an additional 2 times. Cytokinelevels were measured in the sera at the endpoint of the experiment. n=4mice/group. Empty bars=WT; lined bars=KO; white bars=untreated; purplebars=DSS; red bars=DSS+NCK56; green bars=DSS+NCK2187; bluebars=DSS+SlpA. Data are shown as mean±SEM. *P<0.05, **P<0.01,***P<0.001.

FIGS. 10A-10B. Dextran sodium sulfate (DSS) treatment and oral gavageregimens. (A) DSS-induced colitis prevention study; mice were orallygavaged with NCK56, NCK2187 or SlpA at the specified time points. 3% DSSwas given in the drinking water from days 0 to 5. (B) DSS-inducedcolitis therapy. 3% DSS was given in the drinking water from days 0 to5. Mice were then orally gavaged with NCK56, NCK2187, or SlpA at thecircled time points.

FIGS. 11A-11E. L. acidophilus-SlpA protects against DSS-induced colitis.C57BL/6 mice were orally gavaged with NCK56, NCK2187 or SlpA, and 3% DSSwas given in the drinking water. Colitis development in the mice wasmonitored by measuring weight loss (A), diarrhea development (B),presence of fecal occult blood (FOB) (C), gross morphology of the colons(D), and histopathology (E). *P<0.05, **P<0.01 and ***P<0.001representing the specified group by its color compared with PBS.

FIGS. 12A-12E. L. acidophilus-SlpA reverses DSS-induced colitis. C57BL/6mice were given 3% DSS in the drinking water, and were then orallygavaged with NCK56, NCK2187 or SlpA. Colitis development in the mice wasmonitored by measuring weight loss (A), diarrhea development (B),presence of fecal occult blood (FOB) (C), gross morphology of the colons(D), and histopathology (E). *P<0.05 and **P<0.01 representing thespecified group by its color compared with PBS.

FIGS. 13A-13E. L. acidophilus-SlpA protects against pathogenic Tcell-induced colitis. Rag^(1−/−) mice were injected with 10⁶ CD4⁺CD25⁻ Tcells, and were then orally gavaged with NCK56, NCK2187, or SlpA, 1 and3 days after transfer, and subsequently once a week for 4 consecutiveweeks. Colitis development in the mice was monitored by measuring weightloss (A), diarrhea development (B), presence of fecal occult blood (FOB)(C, gross morphology of the colons (D), and histopathology (E). *P<0.05,**P<0.01 and ***P<0.001 representing the specified group by its colorcompared with PBS.

FIGS. 14A-14B. NCK2187 induces the generation of Tregs in healthycontrols. C57BL/6 mice were orally gavaged with 10⁹ CFU NCK56 (WT),NCK2187 (SlpA⁺), or left untreated; immune responses were analyzed byflow cytometry 3 days-post gavage. When compared to untreated orNCK56-treated mice, NCK2187 led to an expansion of total Tregs in themesenteric lymph nodes (MLN; FIG. 14A) and spleens (FIG. 14B) of themice tested. **P<0.01 and ***P<0.001 compared with PBS.

FIGS. 15A-15C. L. acidophilus-SlpA isolation by NaCl. L.acidophilus-SlpA was isolated and purified using NaCl. A. SDS-PAGEcontaining 2.5 μg of LiCl- and NaCl-isolated SlpA stained with COOMASSIEBLUE to visualize the purified protein. B-C. Mass spectrometry dataanalyzed on the Scaffold (Searle 2010) platform showed 97 unique spectrawith 55 unique peptides with the possibility of two proteins (B). Thepredicted protein gi|58336516 (SlpA, SEQ ID NO: 4) shows 54% coveragewhereas gi|362076610 (SlpB, SEC) ID NO: 56) reveals only 18% of coverage(highlighted portion. C). The regions of SipB matching the generatedpeptides are common between SlpA and SlpB (shown in the red box, C), andno single unique peptide from SlpB was identified.

FIGS. 16A-16E. NaCl-purified SlpA is not toxic to mice. A-E. C57BL/6mice were treated orally every other day with SlpA (0, 150, 300, 600μg/100 μL per mouse), for a total of four times. One-week later, micewere sacrificed and a whole blood chemistry profile was generated foreach mouse with a comprehensive metabolic chemistry panel, using aVetScan V2S analyzer. All animal experiments were performed under theguidelines of the Animal Welfare Act and the Public Health Policy onHumane Care, and with approval by the Institutional Animal Care and UseCommittee (IACUC protocol 201406559) at the University of Florida.

FIGS. 17A-17E. Generated mAb BM1 recognizes L. acidophilus-SlpA. C57BL/6mice were immunized once a week for 3 months with 100 μg of SlpA, and300 μg of heat-killed Lactobacillus gasseri as adjuvant. Polyclonal serawere tested for recognition of isolated SlpA by Western Blot (WB), andsplenic cells from SlpA-reactive mice were fused with Sp2/0 myelomacells at a ratio of 7:1. Hybridomas were seeded on semi-solid medium forclone selection and screening. Subsequently, clones were screened byELISA for SlpA reactivity. Reactive clones were isotyped and all IgMsecretors removed. Clone BM1 (IgG) was selected for its ability torecognize SlpA by WB (A), flow cytometry (B), confocal microscopy (C),and ELISA (D, E). A. L. acidophilus-SlpA detection by WB with BM1. 100ng of purified SlpA, 10⁸ CFU L. acidophilus (L. a.), 10⁸ CFU L. reuteri(L. r.), or 100 ng of BSA. Proteins separated by SDS-PAGE weretransferred onto a PVDF membrane and detected by BM1. B. L.acidophilus-SlpA detection with BM1 by flow cytometry. CarboxylatedDynabeads were coated with purified SlpA and reactivity of BM1 mAbconfirmed by Canto II flow cytometry. Data were analyzed by FlowJo.Experiments were performed at least three times with similar trends. C.L. acidophilus-SlpA detection with BM1 by confocal microscopy. RAW 264.7cells were pulsed for 1 or 3 hrs with NaCl purified SlpA (10 μg/mL).Subsequently, cells were fixed and stained with BM1 mAb for detection byconfocal microscopy. Cells were incubated with BM1 mAb, overnight. Cellswere washed and subsequently incubated with a secondary antibody (ALEXAFLUOR 488 anti-mouse IgG1, 1:100) for 4 hrs. Nuclei were stained withDAPI (15 min) and visualized by a Zeiss confocal microscope. D. L.acidophilus-SlpA detection with BM1 by ELISA. ELISA plates were coatedwith 500 ng of purified SlpA overnight, and binding by BM1 was testedthereafter. E. Germ-free (GF) mice were orally treated with 10⁹ CFU L.acidophilus, 150 μg of SlpA, or left untreated. Fecal pellets from thesemice were used to coat ELISA plates; BSA was used as a negative control.BM1 by ELISA. ELISA plates were coated with 500 ng of purified SlpAovernight, and binding by BM1 was tested thereafter. E. Germ-free (GF)mice were orally treated with 10⁹ CFU L. acidophilus, 150 μg of SlpA, orleft untreated. Fecal pellets from these mice were used to coat ELISAplates; BSA was used as a negative control. BM1 mAb only bound to platescoated with feces derived from treated mice. All animal experiments wereperformed under the guidelines of the Animal Welfare Act and the PublicHealth Policy on Humane Care, and with approval by the InstitutionalAnimal Care and Use Committee (IACUC protocol 201406559) at theUniversity of Florida. ***denotes statistical significance p<0.01,***p<0.001.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1: Amino acid sequence of phosphoglycerol transferase proteinfrom L. acidophilus (Genbank Accession No. AAV42337.1).

SEQ ID NO: 2: Amino acid sequence of SlpB protein from L. acidophilus(Genbank Accession No. YP_193105).

SEQ ID NO: 3: Amino acid sequence of SlpX protein from L. acidophilus(Genbank Accession No. YP_193425).

SEQ ID NO: 4: Amino acid sequence of SlpA protein from L. acidophilus(Genbank Accession No. P35829).

SEQ ID NO: 5 to 46: The list of primer sequences for Real-Time PCRanalyses (see Table 1).

SEQ ID NO: 47 to 55: SlpA protein sequences as discussed below.

DETAILED DISCLOSURE OF THE INVENTION

The current invention provides a bacterium lacking on its surface LTA,SlpB and SlpX. The current invention also provides SlpA as an effectormolecule expressed by bacteria, for example, L. acidophilus and its invivo protective role in inflammation mediated diseases (inflammatorydiseases), for example, inflammation mediated diseases of thegastrointestinal tract such as IBD or other inflammatory diseases, suchas allergies, ankylosing spondylitis, Crohn's disease, diabetes, Type Idiabetes, gastroesophageal reflux disease, Hashimoto's thyroiditis,hyperthyroidism, hypothyroidism, Irritable Bowel Syndrome (MS),interstitial cystitis (IC), Lofgren's syndrome, lupus erythematosis,myasthenia gravis, multiple sclerosis, osteoarthritis, polymyalgiarheumatica, prostatitis, psoriasis, psoriatic arthritis, Raynaud'ssyndrome/phenomenon, reactive arthritis (Reiter syndrome), restless legsyndrome, reflex sympathetic dystrophy (RSD), rheumatoid arthritis,scleroderma, Sjögren's syndrome, ulcerative colitis and uveitis.

A bacterium lacking LTA and methods of preparing such bacterium aredescribed in US Patent Application Publication 20130224153, the contentsof which are incorporated by reference herein in its entirety,particularly, paragraphs [0031] to [0034]. In addition to modificationsrequired to decrease or eliminate display of LTA on the surface, thecurrent invention provides a bacterium further modified to decrease oreliminate the display of SlpB and SlpX on the surface. In someembodiments, bacterial cells lacking LTA expression on the cell surfaceare genetically modified to decrease or eliminate SlpB and SlpXexpression on the cell surface. Such cells can be genetically modifiedto expression SlpA and used in the methods disclosed herein. Yet otherembodiments utilize bacterial cells genetically modified to express SlpAbut which lack LTA expression on the cell surface and also lack genesencoding SlpB and SlpX or orthologs of SlpB and SlpX (i.e., proteinhomologs that are present within different species and have very similaror identical function). Non-limiting examples of such bacterial cellsinclude those that lack genes encoding phosphoglycerol transferaseprotein (Genbank Accession No. AAV42337.1; SEQ ID NO: 1), SlpB and SlpXor orthologs of phosphoglycerol transferase (Genbank Accession No.AAV42337.1), SlpB and SlpX. For example, the cells do not express SEQ IDNO: 2 or the SlpB polypeptides associated with Uniprot access numbersQ48508, C2HR61, Q5FMK0, Q8GFE5, J9W284, J9W905, B1H0V4, H6VTN5, Q09FL7,V7HZR4, S4NDQ7, S4NKH4, S4NL65 and S4NQU9 and SEQ ID NO: 3 or the SlpXpolypeptides associated with SlpX protein are provided by Uniprot accessnumbers C2HMW6, S6E4Y8, S6DRU6, S6DL03, S6E593, S6DQJ3, FOTJ46, Q5FLNO,D4YUC6, R5ZGF3, E4SM72, F2M2V8, C2KB60, D5H1S0, I7KQ44, U6FUJ7, U6FJC0,U6F914, U6F7V6, U6F834, U4QN79, U4QA33, F3MP54, F0NWR2, F0NVR1, F6CEM8,F6CBQ1, I7JYF2 and C2ELK0.

In certain embodiments, the phosphoglycerol transferase proteincomprises SEQ ID NO: 1, SlpB protein comprises the amino acid sequenceof SEQ ID NO: 2 and SlpX protein comprises the amino acid sequence ofSEQ ID NO: 3. Accordingly, in addition to the modifications required toreduce the surface display of LTA, the bacterium of the currentinvention has been further genetically modified to decrease or eliminatethe expression of a polypeptide comprising amino acid sequence of SEQ IDNO: 1, comprising amino acid sequence of SEQ ID NO: 2 and a polypeptidecomprising the amino acid sequence of SEQ ID NO: 3. In an embodiment, inaddition to the modifications required to reduce or eliminate thesurface display of LTA, the bacterium of the current invention hasdecreased or eliminated expression of a polypeptide comprising the aminoacid sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99%sequence identity to the amino acid sequence of SEQ ID NO: 1, at least70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acidsequence of SEQ ID NO: 2 and a polypeptide comprising the amino acidsequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequenceidentity to the amino acid sequence of SEQ ID NO: 3.

Certain examples of phosphoglycerol transferase protein from L.acidophilus (Genbank Accession No. AAV42337.1) protein are provided byUniprot access numbers Q5FLT7, A0A0D5MGR2, F3MQQ2, U6F845, U6FPM2,U6FK51, A8YTT6, U6F7B9, C7XM54, E3R4H1, C2KGR7, D0DKS2 and K1NT37 (eachof which is hereby incorporated by reference in their entireties).Additional examples of phosphoglycerol transferase proteins are wellknown to a person of ordinary skill in the art and such embodiments arewithin the purview of the current invention.

Certain examples of SlpB protein are provided by Uniprot access numbersQ48508, C2HR61, Q5FMK0, Q8GFE5, J9W284, J9W905, B1H0V4, H6VTN5, Q09FL7,V7HZR4, S4NDQ7, S4NKH4, S4NL65 and S4NQU9 (each of which is herebyincorporated by reference in their entireties). Additional examples ofSlpB proteins are well known to a person of ordinary skill in the artand such embodiments are within the purview of the current invention.

Certain examples of SlpX protein are provided by Uniprot access numbersC2HMW6, S6E4Y8, S6DRU6, S6DL03, S6E593, S6DQJ3, F0TJ46, Q5FLNO, D4YUC6,R5ZGF3, E4SM72, F2M2V8, C2KB60, D5H1S0, I7KQ44, U6FUJ7, U6FJC0, U6F914,U6F7V6, U6F834, U4QN79, U4QA33, F3MP54, F0NWR2, F0NVR1, F6CEM8, F6CBQ1,I7JYF2 and C2ELK0 (each of which is hereby incorporated by reference intheir entireties). Additional examples of SlpX proteins are well knownto a person of ordinary skill in the art and such embodiments are withinthe purview of the current invention.

The genetic modifications resulting in decreased or eliminatedexpression of the polypeptides include, but are not limited to, deletingthe entire coding region of the gene or a portion of the codingnucleotide sequence, introducing a frame shift mutation, a missensemutation, an insertion, by introducing a stop codon or a combinations ofany of the aforementioned mutations. Additional mutations which wouldlead to decreased, or eliminated, expression of a polypeptide ofinterest and methods of introducing such mutations into a bacterium arewell known to a person of ordinary skill in the art and such embodimentsare within the purview of the claimed invention. In one embodiment ofthe invention, the upp-counterselective knockout strategy (described inGoh et al., 2009) which is hereby incorporated by reference in itsentirety) was used to generate in-frame deletions in the slpB and slpXgenes of L. acidophilus NCK2030 to produce L. acidophilus NCK2187.

In a certain embodiment, the lactic acid bacterium is a surface layerprotein expressing Lactobacillus. These bacterial cells may also bereferred to as probiotic bacterial cells. Non-limiting examples of suchlactic acid bacteria include, but are not limited to, L. acidophilus, L.amylolyticus, L. amylovorus, L. brevis, L. brevis ssp gravesensis, L.buchneri, L. crispatus, L. gallinarum, L. gigeriorum, L.helveticus/suntoryeus, L. hilgardii, L. kefiranofaciens, L. pasteurii,L. lactis and L. ultunensis.

An embodiment provides a composition comprising the recombinantbacterium of the current invention and a pharmaceutically acceptablecarrier and/or excipient.

The bacteria of the current invention provide protective role ininflammation mediated diseases, for example, inflammation mediateddiseases of gastrointestinal tract such IBD. Accordingly, certainembodiments of the current invention provide methods of treating and/orpreventing an inflammation mediated disease of the gastrointestinalsystem in a subject, the method comprising, administering to the subjecta therapeutically effective amount of the bacterium of the currentinvention. In one embodiment, the bacterium is orally administered tothe subject.

In certain embodiments of the invention a subject is a mammal.Non-limiting examples of a mammal treatable according to the methods ofthe current invention include mouse, rat, dog, guinea pig, cow, horse,cat, rabbit, pig, monkey, ape, chimpanzee, and human. Additionalexamples of mammals treatable with the methods of the current inventionare well known to a person of ordinary skill in the art and suchembodiments are within the purview of the current invention.

For the purposes of the current invention, a probiotic food refers to afood which contains microorganisms associated with beneficial effects tohumans and animals upon ingestion of the probiotic food. Non-limitingexamples of probiotic food include yogurt, fermented vegetable, kefir,sauerkraut, miso soup, pickle, tempeh and kimchi.

For the purposes of this invention the term “inflammation mediateddisease” or “inflammatory disease” refers to a disease characterized bya dysregulation of the normal immune response. Inflammation mediateddiseases (inflammatory diseases) can cause organ damage, and areassociated with increased morbidity and/or mortality. An example ofimmune dysregulation is the inappropriate activation of inflammatorycytokines, such as IL-12, IL-6 or TNF alpha, whose actions lead topathological consequences.

For the purposes of this invention the terms “treatment, treating,treat” or equivalents of these terms refer to healing, alleviating,relieving, altering, remedying, ameliorating, improving, or affectingthe condition or the symptoms of a subject suffering with a disease, forexample, a gastrointestinal disorder. The subject to be treated can besuffering from or at risk of developing the disorder, for example, agastrointestinal disorder, including, for example, an IBD or be at riskof developing an IBD. When provided therapeutically, the bacterium isprovided at (or shortly after) the onset of a symptom. The therapeuticadministration of the substance serves to attenuate any actual symptom.

For the purposes of this invention, the terms “preventing, preventive,prophylactic” or equivalents of these terms are indicate that therecombinant bacterium is provided in advance of any disease symptoms andare a separate aspect of the invention (i.e., an aspect of the inventionthat is distinct from aspects related to the terms “treatment, treating,treat” or equivalents of these terms which refer to healing,alleviating, relieving, altering, remedying, ameliorating, improving, oraffecting the condition or the symptoms of a subject suffering with aninflammatory disease, for example, a gastrointestinal disorder). Theprophylactic administration of the recombinant bacterium serves toprevent or attenuate any subsequent symptoms or disease.

By “therapeutically effective dose,” “therapeutically effective amount”,or “effective amount” is intended to be an amount of a recombinantbacterium disclosed herein or the amount of SlpA that, when administeredto a subject, decreases the inflammatory response, or reduces anyincrease in an inflammatory response as compared to untreated subjects.“Positive therapeutic response” refers to, for example, improving thecondition of at least one of the symptoms of an inflammatory disorder.

An effective amount of the therapeutic agent is determined based on theintended goal. The term “unit dose” refers to a physically discrete unitsuitable for use in a subject, each unit containing a predeterminedquantity of the therapeutic composition calculated to produce thedesired response in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject and theprotection desired. Precise amounts of the therapeutic composition alsodepend on the judgment of the practitioner and are peculiar to eachindividual. Generally, the dosage of recombinant bacteria will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. In specificembodiments, it may be desirable to administer the bacterium in therange of about 10⁴ to about 10¹² CFU, 10⁵ to 10¹¹ CFU, 10⁶ to 10¹⁰ CFU,10⁸ to 10¹⁰ CFU or 10⁸ to 10¹² CFU.

In some embodiments of the invention, the method comprisesadministration of multiple doses of the bacterium. The method maycomprise administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, or more therapeutically effective doses of a compositioncomprising the bacterium as described herein. In some embodiments, dosesare administered over the course of 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days, or more than30 days. The frequency and duration of administration of multiple dosesof the compositions is such as to reduce or prevent an inflammatoryresponse and thereby treat or prevent a gastrointestinal disorder.Moreover, treatment of a subject with a therapeutically effective amountof the recombinant bacterium of the invention can include a singletreatment or can include a series of treatments. It will also beappreciated that the effective dosage of a bacterium used for treatmentmay increase or decrease over the course of a particular treatment.Changes in dosage may result and become apparent from the results ofdiagnostic assays for detecting inflammation known in the art anddescribed herein.

The present invention also includes combinations of the recombinantbacteria with one another, and/or with one or more other agents usefulin the treatment of an inflammation mediated disease of the GI tract.For example, bacteria of the invention may be administered incombination with effective doses of conventional anti-inflammatoryagents, such as sulfasalazine, cyclosporine, prednisone,methylprednisone, budesonide, mesalamine, azathioprine, TNF inhibitors,methotrexate, or 6-mercaptopurine, a corticosteroid, infliximab orcombinations thereof, for treatment of inflammation mediated diseases ofthe GI tract. The term “administration in combination” refers to bothconcurrent and sequential administration of the active agents. Thecombination therapies are of course not limited to the agents providedherein, but include any composition for the treatment of inflammatorydisorders.

In certain embodiments, the inflammation mediated disease treatedaccording to the current invention is IBD. Non-limiting examples of IBDinclude Crohn's disease or ulcerative colitis. Additional examples ofIBD are well known to a person of ordinary skill in the art and suchembodiments are within the purview of the current invention. Asdiscussed above, the disclosed methods and compositions are aimed atimproving the condition of at least one of the symptoms of aninflammatory disorder, such as IBD.

The current invention also provides SlpA as an effector moleculeexpressed by bacteria and which provide protective role in inflammationmediated diseases, for example, inflammation mediated diseases ofgastrointestinal tract such IBD. Accordingly, certain embodiments of thecurrent invention provide an isolated bacterial SlpA protein or anon-naturally occurring protein derivative thereof.

An example of bacterial SlpA protein is provided by a protein having thesequence of SEQ ID NO: 4 or a protein having at least 95% sequenceidentity to the sequence of SEQ ID NO: 4. Certain other examples of SlpAprotein are provided by Uniprot access numbers C2HR60 (SEQ ID NO: 47),P35829 (SEQ ID NO: 48), G1UE81 (SEQ ID NO: 49), Q9Z4J9 (SEQ ID NO: 50),H6VTN4 (SEQ ID NO: 51), Q09FM2 (SEQ ID NO: 52), L7YE91 (SEQ ID NO: 53),K8DVK7 (SEQ ID NO: 54) and F0NUB7 (SEQ ID NO: 55) (each of which ishereby incorporated by reference in its entirety). Additional examplesof SlpA proteins are well known to a person of ordinary skill in the artand such embodiments are within the purview of the current invention foruse in formulation of the compositions disclosed herein as well as themethods of using such compositions for the treatment of inflammatorydiseases.

For the purposes of this invention a “non-naturally” occurring proteinderivative indicates that the protein derivative is different than theits naturally occurring counterpart in some manner. Certain examples ofmodifications which can distinguish a non-naturally occurring proteinderivative from its naturally occurring counterpart include mutations inthe amino acid sequences (e.g., point mutations or the introduction ofone or more glycosylation site into the protein), non-naturallyoccurring post-translational modifications (e.g. glycosylation orphosphorylation patterns), attachment to the protein of extraneousmolecules (e.g. molecular labels, such as radioisotopes or fluorescentlabels, polyethyleneglycol (PEG), etc.). Additional examples of suchmodifications are well known to a person of ordinary skill in the artand such embodiments are within the purview of the current invention.

In one embodiment, the non-naturally occurring SlpA protein derivativeaccording to the current invention comprises a molecular labelconjugated to a bacterial SlpA protein, for example, SlpA protein havingthe sequence of SEQ ID NO: 4 or the protein having at least 95% sequenceidentity to the sequence of SEQ ID NO: 4. The label can be a radiolabel,fluorescent label, affinity label, targeting label.

In another embodiment, the non-naturally occurring SlpA proteinderivative according to the current invention comprises a protein havingone or more mutations in the naturally occurring sequence of a bacterialSlpA protein, for example, SlpA protein having a sequence of SEQ ID NO:4 or having a sequence at least 95% identical to the sequence of SEQ IDNO: 4. In certain embodiments, the non-naturally occurring SlpA proteinderivative comprises about 1 to about 20 mutations, about 3 to about 15mutations, or about 5 to about 10 mutations. In another embodiment, themutations do not negatively affect the ability of the non-naturallyoccurring SlpA protein derivative of the current invention of protectingagainst inflammation mediated diseases, for example, inflammationmediated diseases of gastrointestinal tract such IBD.

An embodiment of the current invention also provides a compositioncomprising the non-naturally occurring SlpA protein derivative and apharmaceutically acceptable carrier and/or excipient.

The pharmaceutically acceptable carrier and/or excipient comprisesubstances, such as an inert vehicle, or pharmaceutical acceptableadjuvants, preservatives etc. Examples pharmaceutically acceptablesubstances are well known to a person of ordinary skill in the art andsuch embodiments are within the purview of the current invention.

The pharmaceutical composition may be a liquid formulation or a solidformulation. When the pharmaceutical composition is a solid formulationit may be formulated as a tablet, a sucking tablet, a chewing tablet, achewing gum, a capsule, a sachet, a powder, a granule, a coatedparticle, a coated tablet, an enterocoated tablet, an enterocoatedcapsule, a melting strip or a film. When the pharmaceutical compositionis a liquid formulation it may be formulated as an oral solution, asuspension, an emulsion or syrup. Said composition may further comprisea carrier material independently selected from, but not limited to, thegroup consisting of lactic acid fermented foods, fermented dairyproducts, resistant starch, dietary fibers, carbohydrates, proteins, andglycosylated proteins.

Pharmaceutical compositions, as disclosed herein, can be formulated inaccordance with standard pharmaceutical practice (see, e.g., Remington:The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro,Lippincott Williams & Wilkins, 2000 and Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, New York) known by a person skilled in the art. Pharmaceuticalcomposition according to the invention may also be formulated to releaseactive agents substantially immediately upon administration or at anypredetermined time or time period after administration.

For oral administration, the composition can be formulated intoconventional oral dosage forms such as tablets, capsules, powders,granules and liquid preparations such as syrups, elixirs, andconcentrated drops. Non-toxic solid carriers or diluents may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,glucose, sucrose, magnesium, carbonate, and the like. For compressedtablets, binders, which are agents which impart cohesive qualities topowdered materials are also necessary. For example, starch, gelatin,sugars such as lactose or dextrose, and natural or synthetic gums can beused as binders. Disintegrants are also necessary in the tablets tofacilitate break-up of the tablet. Disintegrants include starches,clays, celluloses, algins, gums and cross-linked polymers. Moreover,lubricants and glidants are also included in the tablets to preventadhesion to the tablet material to surfaces in the manufacturing processand to improve the flow characteristics of the powder material duringmanufacture. Colloidal silicon dioxide is most commonly used as aglidant and compounds such as talc or stearic acids are most commonlyused as lubricants.

Pharmaceutical composition can also be formulated as a food composition,a dietary supplement, a functional food, a medical food or a nutritionalproduct as long as the required effect is achieved, i.e. treatmentand/or prevention of an inflammatory disorder of the gastrointestinaltract. Said food composition may be chosen from the group consisting ofbeverages, yogurts, juices, ice creams, breads, biscuits, crackers,cereals, health bars, spreads and nutritional products. The foodcomposition may further comprise a carrier material, wherein saidcarrier material is chosen from the group consisting of lactic acidfermented foods, fermented dairy products, resistant starch, dietaryfibers, carbohydrates, proteins and glycosylated proteins.

Accordingly, the current invention provides a method of treating orpreventing an inflammation mediated disease of the gastrointestinalsystem in a subject, the method comprising, administering to the subjecta therapeutically effective amount of the composition comprising theSlpA protein or a non-naturally occurring SlpA derivative. In certainembodiments, the composition is orally administered to the subject. SlpAprotein or a non-naturally occurring SlpA derivatives can beadministered or formulated in combination with effective doses ofconventional anti-inflammatory agents, such as sulfasalazine,cyclosporine, prednisone, methylprednisone, budesonide, mesalamine,azathioprine, TNF inhibitors, methotrexate, or 6-mercaptopurine, acorticosteroid, infliximab or combinations thereof, for treatment ofinflammation mediated diseases of the GI tract. Non-limiting examples ofsuch diseases include IBD, for example, Crohn's disease or ulcerativecolitis.

Yet another aspect of the invention relates to a method of purifyingSlpA comprising growing SlpA expressing bacterial cells, pelleting saidbacterial cells from culture medium containing said bacterial cells,resuspending said bacterial cells in 5M NaCl for a period of 5 minutesto 24 hours, preferably between 30 minutes and two hours, to form anextraction composition, removing bacterial cells from said extractioncomposition by centrifugation to form a composition containing SlpA anddialyzing or filtering said composition using a dialysis bag orultrafiltrating device having a molecular weight cut-off of 30 kDa toreduce the salt content of said composition containing SlpA. The methodcan further comprise the precipitation of SlpA protein in said dialyzedcomposition comprising SlpA (for example, with 1M NaCl or anothersuitable preciptitating agent). The method can also further comprise thewashing of said precipitated SlpA with water or a buffer andlyophilization of said washed SlpA.

In some embodiments of the purification methodology, the SlpA expressingbacterial cells do not express LTA, SlpB or orthologs thereof or SlpX ororthologs thereof. Alternatively, the SlpA expressing bacterial cells donot express phosphoglycerol transferase or orthologs thereof, SlpB ororthologs thereof or SlpX or orthologs thereof.

Thus, the bacterial cells, in some embodiments: a) express a proteinthat has the amino acid sequence of SEQ ID NO: 4 or the protein has theamino acid sequence at least 70%, 80%, 90%, 95%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 4; and b) do not express (i) apolypeptide comprising the amino acid sequence of SEQ ID NO: 1 or apolypeptide having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99%sequence identity to the amino acid sequence of SEQ ID NO: 1; (ii) apolypeptide comprising the amino acid sequence of SEQ ID NO: 2 or apolypeptide comprising the amino acid sequence having at least 70%, 80%,90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequenceof SEQ ID NO: 2; and (iii) a polypeptide comprising the amino acidsequence of SEQ ID NO: 3 or a polypeptide having at least 70%, 80%, 90%,95%, 97%, 98%, or 99% sequence identity to the amino acid sequence ofSEQ ID NO: 3. In certain aspects of the invention, these bacterial cellsare Lactobacillus strain, such as a Lacotbacillus strain is selectedfrom the group consisting of L. acidophilus, L. amylolyticus, L.amylovorus, L. brevis, L. brevis ssp gravesensis, L. buchneri, L.crispatus, L. gallinarum, L. gigeriorum, L. helveticus/suntoryeus, L.hilgardii, L. kefiranofaciens, L. pasteurii, L. lactis and L.ultunensis.

Materials and Methods

Mice

C57BL/6 (B6), and B6 recombination-activating gene 1-deficient(Rag1^(−/−)) mice were purchased from Jackson Laboratories (Bar Harbor,Me.). Germ-free (GF) B6 mice were obtained from the National GnotobioticRodent Resource Center at the University of North Carolina andmaintained in the GF facilities at the University of Florida (UF). Themouse strain 031934-UCD, C57BL/6-Cd209d^(tm1.1Cfg)/Mmucd (Signr3^(−/−))was provided by the NIH-sponsored Mutant Mouse Regional Resource Center(MMRRC) National System and was backcrossed at the Max Planck Instituteof Colloids and Interfaces, Berlin, Germany. Genotyping of the Signr3gene in WT and Signr3^(−/−) mice was performed according to a protocolprovided by the Consortium for Functional Glycomics. Dr. L. Morel (UF)contributed the FoxP3-GFP mice. Mice were bred in-house in the animalfacility at the College of Veterinary Medicine, UF. Mice were maintainedunder specific pathogen-free, Helicobacter-free conditions and used at6-8 weeks of age in accordance with the Animal Welfare Act and thePublic Health Policy on Humane Care. Procedures were approved by UF'sInstitutional Animal Case and Use Committee (IACUC).

Bacterial Strains

The upp-counterselective knockout strategy was used to generate anin-frame deletion of the phosphoglycerol transferase gene within NCK2030(LTA⁺ SlpB⁻ SlpX⁻ SlpA⁺), resulting in the generation of NCK2187(LTA⁻SlpB⁻SlpX⁻SlpA⁺) (Goh et al., 2009). Wild-type L. acidophilus NCFM(NCK56), and NCK2187 were propagated anaerobically in MRS broth (Difco,BD, Franklin Lakes, N.J.) at 37° C. for 15 hrs. In preparation for oraltreatment, bacteria were washed twice with sterile PBS, and the numberof colony-forming units (CFU) were estimated by measuring the opticaldensity at 600 nm. The concentration of each L. acidophilus strain wasaccordingly adjusted. To determine the clearance kinetics of thedifferent L. acidophilus strains, groups of mice (n=3) were orallygavaged with erythromycin-resistant (Em^(r)) NCK56 or NCK2187 (1×10⁹CFU/100 μL/mouse). Fecal pellets were collected before gavage and everyday thereafter for up to 7 days. Each fecal pellet was then resuspendedin 10% MRS (0.2 g/2 mL). The homogenized material was serially dilutedand plated onto MRS agar containing Em (2 μg/mL). The daily averageexcreted L. acidophilus was quantified. For the oral gavage of mice,each mouse received either 1×10⁹ CFU of NCK56 or NCK2187 in 100 μL ofPBS. Mice enrolled in steady-state studies were orally gavaged every 3days for a total of 4 times, and immune changes analyzed at day 14. Thegavage schedule was determined based on the clearance kinetics of thebacterial strains.

Surface Layer Protein A Isolation

SlpA was purified from NCK2187 with LiCl. Cultures from 18 h grownNCK2187 were pelleted at 5,000 rpm for 10 min. Bacterial pellets werewashed with cold PBS and re-pelleted before extraction. Pellets wereresuspended in 5 M LiCl or 5M NaCl (Sigma-Aldrich, St. Louis, Mo.),gently stirred for 30 min, and the bacteria removed by centrifugation(13,000 rpm, 15 min). Supernatants were dialyzed against distilled waterovernight using a dialysis bag with a cut-off molecular weight of 30 kDafor salt removal. The protein precipitate was dissolved in 1 M LiCl or1M NaCl and pelleted at 13,000 rpm for 30 min. The SlpA proteinpreparation was washed with water a minimum of three times beforelyophilization (FreeZone, Labconco, Kansas City, Mo.). Freeze-dried SlpAwas stored at 4° C. until used. SDS-PAGE gels and proteomics analyseswere used to confirm SlpA purity. For oral gavage, mice were given 150μg of SlpA in 300 μL PBS.

Lamina Propria Leukocyte (LPL) Preparation

Colonic lamina propria cells were isolated, as previously described(Lightfoot et al., 2014). Freshly isolated colons were cut into 0.5 cmsections and intraepithelial lymphocytes removed with a digestion bufferconsisting of HBSS (GIBCO, Life Technologies, Grand Island, N.Y.)containing 5 mM EDTA (GIBCO, Life Technologies) and 10 mM HEPES (GIBCO,Life Technologies), for (20 min, 37° C.). Remaining colon tissues weredigested in DMEM (GIBCO, Life Technologies) supplemented with 0.25 ng/mLCollagenase Type VII (Sigma-Aldrich), 0.125 U/mL Liberase TM ResearchGrade (Roche Applied Science, Indianapolis, Ind.), 10 mM HEPES, 0.1 MCaCl2 (Sigma-Aldrich), and 5% FBS (GIBCO, Life Technologies). Threedigestions of 10 min each at 37° C. were performed. Single cellsuspensions obtained were combined and stained for flow cytometry-basedanalyses or used for ex vivo studies.

Ex Vivo Stimulation of Colonic LPLs

Isolated colonic LPLs were co-cultured with NCK56 or NCK2187 for 12 h at37° C. Supernatants were then collected and stored at −80° C. for latercytokine analyses using Bio-Plex Pro Mouse Cytokine Immunoassay kits(Bio-Rad, Hercules, Calif.). Activation phenotypes of DCs were analyzedby flow cytometry using the appropriate antibodies to quantifyexpression levels of MHC-II molecules and costimulatory markers.

Flow Cytometry and Antibodies

Colonic LPLs were stained as described previously (Lightfoot et al.,2014). Colonic LPLs were stained with LIVE/DEAD Aqua Dead Cell Stain Kit(Molecular Probes, Life Technologies). Washed cells were incubated withMouse Fc Blocking Reagent (Miltenyi Biotec, Auburn, Calif.) per themanufacturer's instructions before staining with combinations of thefollowing antibodies or their corresponding isotype controls: CD45(30-F11), CD11c (N418), CD11b (M1/70), CD11b (M1/70), F4/80 (BM8), GR1(RB6-8C5), I-A/I-E MHCII (2G9), CD3 (145-2C11), CD4 (RM4-5), CD8(53-607), Pro-IL-1β (NJTEN3)/Rat IgG1, κ, IFNγ (XMG1.2)/Rat IgG1, κ,IL-17A (TC11-18H10.1)/Rat IgG1, κ, IL-10 (JES5-16E3)/Rat IgG2b, κ, LAP(TGF-β1, TW7-16B4)/Mouse IgG1, κ, FoxP3 (FJK-16A)/Rat IgG2a, κ, RORγt(AFKJS-9)/Rat IgG2a, κ. For the detection of intracellular cytokines,cells were fixed and permeabilized with BD Cytofix/Cytoperm (BDBiosciences). Colonic T cells were stimulated with phorbol 12-myristate13-acetate (PMA) (50 ng/mL) and ionomycin (2.5 μg/mL) in the presence ofBrefeldin A (Biolegend) for 2.5 h. The Transcription FactorFixation/Permeabilization kit from eBioscience was used for FoxP3staining. After staining, a BD LSRFortessa (BD Biosciences) cellanalyzer was used to acquire fixed cells. Data were analyzed with FlowJosoftware (Tree Star, Ashland, Oreg.).

Antibodies and their corresponding isotype controls were purchased fromeBioscience (San Diego, Calif.), Biolegend (San Diego, Calif.), BDPharmingen, or R&D Systems (Minneapolis, Minn.).

T Cell-Induced Colitis

In preparation for the adoptive transfer of CD45RB^(hi) CD4⁺ T cellsinto Rag1^(−/−) mice, spleen and mesenteric lymph node (MLN) single cellsuspensions obtained from healthy B6 mice were pooled and incubated inAffiniPure Goat Anti-Mouse IgG (H+L)-coated cell culture plates (JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.) at 37° C. for 1 h.CD4⁺ T cells were isolated from non-adherent cells using the CD4⁺ T cellIsolation Kit II (MACS, Miltenyi Biotec, San Diego, Calif.), andCD25⁺CD4⁺ T cells were then depleted by positive selection (MACS,Miltenyi Biotec). Bound CD25⁺CD4⁺ T cells were collected and injectedinto the regulatory T cells (Tregs) group. The resulting cellsuspensions after negative and positive selection was consistentlycomprised of >98% CD25⁻CD45RB^(h1) CD4⁺ T cells. Rag1^(−/−) mice wereorally gavaged once with NCK56, NCK2187, or SlpA prior to the adoptivetransfer of T cells by intraperitoneal injection (i.p.). One day later,the mice were orally gavaged once more, and once a week for 4consecutive weeks thereafter (FIG. 3A). Colitis progression wasmonitored by determining mouse weight loss, diarrhea development, andfecal occult blood (FOB) presence throughout the study. Stoolconsistency was scored as follows: 0=normal, 2=pasty, 4=watery withperianal staining.

DSS-Induced Colitis

WT and Signr3^(−/−) mice were treated with 3% DSS in the drinking waterfor 5 days (made fresh every 2-3 days) to induce colitis. Mice weremonitored for disease progression through day 10 after treatment asdescribed above. For prevention studies, mice were orally gavaged withNCK56, NCK2187, or SlpA at days −3 and −1, then every other day after 3%DSS treatment, for a total of 5 gavages (2 before, and 3 after 3% DSS).

Histopathology

Colitis scores in T cell- and DSS-induced colitis were determined byhistopathology. Tissues were fixed, sectioned, and stained withhematoxylin and eosin (Histology Tech Services, Gainesville, Fla.).Stained sections were analyzed blindly by a boarded veterinarypathologist. Colitis was graded based on 7 parameters (0-17) aspreviously described (Cheng et al., 2014).

FITC-Dextran Intestinal Permeability Assay

Passive transepithelial absorption of FITC-labeled dextran(Sigma-Aldrich) in vivo was used to determine intestinal barrierfunction as previously described (Napolitano et al., 1996). Mice weregavaged with FITC-dextran, MW 4,000 (60 mg/100 g body weight). Blood wascollected retro-orbitally after proper anesthetization; mice weresacrificed after blood collection. Fluorescence intensity in the serumwas measured with a fluorimeter (485 nm excitation, 519 nm emission).FITC-dextran concentrations in the mouse sera were determined fromstandard curves generated by serial dilution of FITC-dextran using blanksubtraction in the test samples using sera from mice that were notgavaged with the permeability tracer.

Colonoscopy of DSS- and T Cell-Induced Colitis Mice

Macroscopic damage in the colons of Rag1^(−/−), WT, and Signr3^(−/−)mice was visualized with a Multi-Purpose Rigid Telescope attached to aTELE PACK X (Karl Storz-Endoscope, Germany). Mice were fasted for 2-4 h,and subsequently the colons of the living subjects were imaged underappropriate anesthetic conditions.

Real-Time PCR and 16S Ribosomal DNA Sequencing

Colonic tissues from Rag1^(−/−), WT, and Signr3^(−/−) mice were isolatedand processed for changes in gene expression as previously described(Lightfoot et al., 2014). Microbiome analyses were performed on theIllumina Miseq (Illumina, Inc., San Diego) as outlined previously(Lightfoot et al., 2014). Primers used, as well as their sequences, arelisted in the Table 1 below showing the list of primer sequences forReal-Time PCR analyses.

TABLE 1 List of primer sequences for Real-Time PCR analyses SEQ IDGene name Sequence (5′-3′) NO: 111b Forward AAGGAGAACCAAGCAACGAC  5Reverse GAGATTGAGCTGTCTGCTCA  6 Ocln Forward GCTGTGATGTGTGTGAGCTG  7Reverse GACGGTCTACCTGGAGGAAC  8 Cd209a Forward TCTGGA TTCAGT AGCTTCACAGG 9 Reverse GGGTCAGTTCTTGGTAGACATTC 10 Cd209bForward TTGA TGGTCAGCGGCAGCAGG 11 Reverse TCAGCAGGAGCCCAGCCAAGA 12Cd209c Forward CTGGAATGACTCTGTCAATGCC 13 Reverse GCCA TCTGCCTTCA TGCTTCA14 Cd209d Forward GGGCCCAACTGGTCATCATA 15 Reverse AGCGTGTAAAGCTGGGTGAC16 Cd209e Forward CCACA TTCCCCTGGTGTTG 17 Reverse CAGAGGCGACAGAGTCTATCA18 Cd209f Forward CTCTTTGGGCCTCTTTTTGCT 19 Reverse AGTATGCACGAATCCTGGAGA20 Cd209g Forward GGCCTCAGCGATCACAGAAG 21 Reverse ACAACGGCTGTCATTCCATTTA22 Muc2 Forward GTGTGGGACCTGACAATGTG 23 Reverse ACAACGAGGTAGGTGCCATC 24Muc3 Forward GCCGTGAATTGTATGAACGGA 25 Reverse CGCAGTTGACCACGTTGACT A 26Tjp1 Forward AGGACACCAAAGCATGTGAG 27 Reverse GGCATTCCTGCTGGTT ACA 28Tjp2 Forward ATGGGAGCAGTACACCGTGA 29 Reverse TGACCACCCTGTCA TTTTCTTG 30Tjp3 Forward TCGGCATAGCTGTCTCTGGA 31 Reverse GTTGGCTGTTTTGGTGCAGG 32Cldn1 Forward TCCTTGTTCGGCTATGTGTC 33 Reverse GGCATGCACCTAAGAATCAG 34Cldn2 Forward GGCTGTTAGGCACATCCAT 35 Reverse TGGCACCAACATAGGAACTC 36Cldn3 Forward AAGCCGAATGGACAAAGAA 37 Reverse CTGGCAAGTAGCTGCAGTG 38Cldn5 Forward GCAAGGTGTATGAATCTGTGCT 39 Reverse GTCAAGGT AACAAAGAGTGCCA40 Cldn8 Forward GCCGGAATCA TCTTCTTCA T 41Reverse CA TCCACCAGTGGGTTGT AG 42 Hsp25 Forward GGTTGCCCGATGAGTGGTC 43Reverse CTGAGCTGTCGGTTGAGCG 44 Hsp72 Forward CTCCCTCTTGCGTTGCCTC 45Forward ACCCGCAGT AAT AGCCA TCTG 46

SIGNR1 and SIGNR3 Binding Assays

C-type lectin receptors, SIGNR1 and SIGNR3, were fused to the Fc part ofhuman IgG1 (SIGNR1-hFc and SIGNR3-hFc) as previously described (Erikssonet al., 2013). Briefly, the extracellular regions of murine SIGNR1 andSIGNR3 were amplified and ligated into the expression vectorpFUSE-hIgG1-Fc2 (Invivogen, Toulouse, France) for expression in CHO-Scells. Expression in CHO cells was driven by an hEF1-HTLV promoter andsecretion into the culture supernatant was mediated by an external IL2signal sequence (IL2ss). Binding of SlpA-coated beads (Dynabeads MyOneCarboxylic Acid, Life Technologies) to fusion proteins was analyzed byflow cytometry.

Statistical Analyses

Representative data indicate mean±SEM. Significance was determined bytwo-tailed unpaired t-tests for two group comparisons (GraphPad Prismv6.0d for Mac OS X, La Jolla, Calif.). Statistical significance fordifferences in weight loss, diarrhea score, and FOB score was calculatedusing multiple unpaired t-tests correcting for multiple comparisons withthe Holm-Sidak method in Prism v6.0d.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—Nck2187 Promotes Intestinal Immune Regulation in Steady-State

Transient colonization of the colon with NCK2025 (LTA⁻) significantlymitigated chemical and T cell-mediated colitis (Mohamadzadeh et al.,2011). Additionally, NCK2025 significantly abated inflammation-promotingpolyposis in Apc^(lox468)×TS4-Cre mouse model, where protectioncorrelated with the regulation of innate and T cell-induced inflammation(Khazaie et al., 2012). Thus, the controlled inflammation may resultfrom the crosstalk between NCK2025-SlpA and intestinal cells. To testthis hypothesis, the upp-counterselective gene replacement strategy wasused to generate in-frame deletions in the slpB and slpX genes ofNCK2030. The LTA⁻ derivative was created by a deletion of thephosphoglycerol transferase gene (Mohamadzadeh et al., 2011) in NCK2030,resulting in NCK2187, which expresses only SlpA (FIGS. 1A-C).

To demonstrate that the newly generated NCK2187 transiently colonizesthe gut, the clearance kinetics of both the erythromycin-resistant NCK56and NCK2187 strains were determined in C57BL/6 (B6) mice that wereorally treated once with 10⁹ CFU/mouse. Data show that mice cleared bothNCK56 and NCK2187 after 3 days, indicating that the deletion of LTA,SlpB, and SlpX in NCK2187 did not alter its transient passage throughthe GI tract when compared to its WT parent (FIG. 1D).

To investigate the activation of colonic DCs when co-cultured with NCK56or NCK2187, colonic cells were obtained from naïve B6 mice. While suchintestinal cell-bacterial co-cultures did not significantly change theexpression of DC costimulatory molecules (e.g., CD40) (not shown) orIL-10, only NCK56 elevated the levels of IL-1β, IL-6, IL-12, and TNF-α(FIG. 1E).

Next, naïve mice were orally gavaged with NCK56 or NCK2187 and colonicimmune responses analyzed. Treatment with NCK2187 significantlyincreased the frequency of colonic FoxP3⁺ Tregs when compared to bothuntreated (PBS) and NCK56-treated mice (FIG. 2A). Moreover, IL-17A⁺ andIFNγ⁺CD4⁺ T cells were significantly reduced by NCK2187 treatment (FIG.2A). NCK2187-treated FoxP3-GFP mice also exhibited higher numbers ofcolonic IL-10⁺ TGF-β1⁺ Tregs than NCK56-treated and untreated mice(FIGS. 2B, 2C). Collectively, oral treatment with this novel L.acidophilus strain induced colonic regulatory immune responses.

Example 2—Protective Property of Nck2187 and its Slpa AgainstInflammation and Dysbiosis

To elucidate the consequences of the immunoregulatory responses observedabove during inflammation, Rag1^(−/−) mice adoptively transferred withCD45RB^(hi) CD4⁺ T cells were orally treated with NCK56, NCK2187, itspurified SlpA, or PBS (FIG. 3A). Untreated (PBS) and NCK56-treated micewith adoptively transferred T cells developed severe colitis asdemonstrated by weight loss, bloody diarrhea, shortening of the colon,and increased damage of the colon (FIGS. 3A-C, FIG. 8A). Furthermore,the levels of systemically induced proinflammatory IL-1β, IL-6, TNF-α,IFNγ, G-CSF, and MIP-1α were significantly enhanced in the sera of thesegroups of mice (FIG. 3D). In contrast, similar to the Tregco-transferred mice, NCK2187 and its purified SlpA significantlyprotected Rag1^(−/−) mice from T cell-induced colitis (Tables 2-4).

TABLE 2 Statistical analysis of weight loss curves in T cell-inducedcolitis. Weight Loss (p-value) vs. + PBS vs. + NCK56 Week + Tregs +NCK2187 + SlpA + Tregs + NCK2187 + SlpA 1 1.0   1.0   1.0   1.0   1.0  1.0   2 0.0601 0.1714 0.1224 0.5588 0.9927 0.8543 3 0.2331 0.0237 0.19430.5119 0.0556 0.5386 4 0.0653 0.0778 0.1229 0.1194 0.1220 0.2526

TABLE 3 Statistical analysis of diarrhea score curves in T cell-inducedcolitis. Diarrhea Score (p-value) vs. + PBS vs. + NCK56 Week + Tregs +NCK2187 + SlpA + Tregs + NCK2187 + SlpA 1 1.0 1.0 1.0 1.0 1.0 1.0 2 1.01.0 1.0 1.0 1.0 1.0 3 1.0 1.0 1.0 1.0 1.0 1.0 4 0.0231 0.0024 0.00140.2100 0.0592 0.0287 5 0.1139 0.1100 0.507225 0.0101 0.0094 0.0749 60.0011 <0.0001 0.0046 0.0011 <0.0001 0.0031 7 <0.0001 <0.0001 0.0008<0.0001 <0.0001 0.0003

TABLE 4 Statistical analysis of fecal occult blood (FOB) score curves inT cell induced colitis. FOB Score (p-value) vs. + PBS vs. + NCK56 Week +Tregs + NCK2187 + SlpA + Tregs + NCK2187 + SlpA 1 1.0 1.0 1.0 1.0 1.01.0 2 1.0 1.0 1.0 1.0 1.0 1.0 3 1.0 1.0 1.0 1.0 1.0 1.0 4 0.0730 0.01760.2532 0.0721 0.0175 0.2479 5 0.0227 0.0008 0.0733 0.0009 <0.0001 0.00276 0.0014 0.0015 0.0009 0.0001 0.0001 <0.0001 7 <0.0001 <0.0001 <0.0001<0.0001 0.0003 <0.0001

NCK2187- and SlpA-treated mice gained weight throughout the course ofthe study and did not develop significant bloody diarrhea in the waythat the PBS and NCK56 groups did (FIG. 3A). Furthermore, cecal andcolonic atrophy due to pathogenic inflammation was not observed in thesemice, as the tissue destruction and immune cell infiltration associatedwith T cell-induced colitis were significantly abrogated in NCK2187 andSlpA treated groups (FIGS. 3B and 3C). Accordingly, systemicinflammation was significantly reduced in these groups of mice (FIG.3D).

The genes encoding the receptors for LTB₄, Ltb4r1 and Ltb4r2 weresignificantly down-regulated in the colons of NCK2187- and SlpA-treatedmice (FIG. 8B), which may have contributed to the reduced expression ofcolonic Il1b (FIG. 8B).

T cell-induced colitis resulted in intestinal epithelial erosions andulcerations in mice that did not receive NCK2187 or purified SlpA (FIG.3C). Indeed, the colonic expression of tight junction associated geneswas significantly downregulated in PBS- and NCK56-treated Rag1^(−/−)mice (FIG. 4A).

Furthermore, FITC-dextran permeability assays confirmed that these micewere suffering from a dysfunctional intestinal barrier (FIG. 4A).Accordingly, NCK2187 and SlpA significantly protected barrier integrityand function (FIG. 4A). An immunologically and anatomically weakenedintestinal epithelial barrier during acute intestinal inflammationallows luminal bacteria to interact with the intestinal mucosae and theinfiltrating immune cells, initiating inflammatory responses directedagainst the gut commensals and introducing dysbiosis. To elucidate thestatus and composition of the microbiota in the T cell-induced colitismodel (week 7), we analyzed the microbial communities in the colons ofthe different experimental groups and found that the severity of colitiswas associated with significant changes in the microbiota (FIGS. 4B-D,Table 5). UniFrac analyses revealed that fecal bacterial diversity inPBS- and NCK56 treated Rag1^(−/−) mice was modified in such a way thatthese groups were found to cluster separately from each other and fromthe protected mice (FIG. 4B). Conversely, SlpA-, NCK2187-, andTreg-treated groups clustered together and showed similar phyladistributions (FIGS. 4B and 4C). Induced colitis in PBS- andNCK56-treated groups resulted in a significant contraction of members ofthe Bacteroidetes phyla (FIG. 4C). Additionally, the normallyunderrepresented Verrucomicrobia phyla was increased in thesecolitogenic mice (FIG. 4C), suggesting a shift in the intestinal milieuand the substrates available in the inflamed colon, which may promotepreviously underrepresented microbial communities so that they dominatethe population. Alterations in the microbial composition were alsomanifested at lower taxonomic levels: NCK2187-, SlpA-, and Treg-treatedgroups once again showed similar relative abundance and distribution ofseveral unclassified genera (FIG. 4D).

TABLE 5 Analysis of phyla distribution in the fecal microbiota after Tcell-induced colitis. p-value vs. + PBS vs. + NCK56 Mean Value (%)Phylum + Tregs + NCK2187 + SlpA + Tregs + NCK2187 + SlpA + PBS + NCK56 +Tregs + NCK2187 + SlpA Actinobacteria 0.1818 0.1595 0.1812 0.2663 0.21880.2654 1.64 1.16 0.79 0.62 0.80 Bacteroidetes 0.0010 0.0129 0.0276<0.0001 0.0009 0.0029 12.70 7.44 31.32 25.30 24.87 Firmicutes 0.00530.0714 0.1022 <0.0001 0.0010 0.0043 80.43 88.89 66.45 73.35 72.61Proteobacteria 0.2838 0.3294 0.0673 0.1508 0.1364 0.1956 0.14 1.15 0.260.21 0.42 Tenericutes 0.0494 0.4280 0.1102 0.0015 0.1215 0.0208 0.540.24 1.17 0.49 1.22 Verrucomicrobia 0.0244 0.0249 0.0263 0.0796 0.08340.0934 3.79 1.13 0.01 0.06 0.08

Example 3—L. acidophilus-Slpa Binding to Signr3 Promotes ColonicRegulatory Immune Responses

Symbiotic bacteria and their gene products dictate the nature of innateresponses via their sensing receptors (Ivanov and Honda, 2012; Yang etal., 2014); however such stimulatory signals must be regulated by otherreceptors to avoid intestinal inflammation. As previously stated, SIGNR3exhibits the most biochemical similarity to human DC-SIGN. We screenedall known murine SIGNR1-8 and found that Signr1 and Signr3 genes aredifferentially activated in the colonic tissue of mice orally treatedwith NCK2187 (FIG. 5A), prompting us to evaluate the binding of SlpA toSIGNR1 and SIGNR3.

Subsequently, the corresponding extracellular domain of SIGNR1 andSIGNR3 were fused to the Fc portion of human IgG₁ (SIGNR1-hFc,SIGNR3-hFc) and then transiently expressed in Chinese hamster ovary(CHO)-S cells (data not shown). Data demonstrate that while expressedSIGNR3-hFc bound to purified SlpA coated onto charged beads, SIGNR1-hFc,DCAR-hFc (control protein), hFc, and the secondary rat anti-human Fcantibody alone did not, suggesting SlpA-binding specificity to SIGNR3(FIG. 5B).

To clarify the role of SlpA: SIGNR3 binding and signaling in vivo, wefirst orally treated WT and Signr3^(−/−) mice with our bacterial strainsand analyzed the immunologic responses induced in steady-state. WhileNCK2187 treatment led to reduced IL-10 in both conventional (data notshown) and germ-free (GF) B6 mice (FIG. 5C, left), no anti-inflammatoryeffects were observed in NCK2187-treated Signr3^(−/−) mice (FIG. 5C,right). Furthermore, the Treg-inducing properties of NCK2187 (FIGS. 2Aand 2B) were abrogated in Signr3^(−/−) mice (FIG. 5D). These datastrongly suggest that NCK2187 delivers immunoregulatory signals via itsinteraction with SIGNR3.

Example 4—Nck2187 and its Slpa Cannot Prevent Dextran Sulfate Sodium(DSS) Induced Colitis in the Absence of Signr3 Signaling

Previous reports have highlighted the role of specific CLRs inexperimental colitis. For instance, mice lacking Signr1 expression areless susceptible to induced colitis (Saunders et al., 2010), while micedeficient in Dectin1 and Signr3 exhibit exacerbated disease (Eriksson etal., 2013; Iliev et al., 2012). To further investigate SlpA: SIGNR3signaling in disease conditions, DSS-treated WT and Signr3^(−/−) micewere orally gavaged with NCK56, NCK2187, or SlpA, and monitored fordisease progression.

Consistent with the T cell-induced colitis model, disease progressionand severity were significantly reduced in WT mice orally treated withNCK2187 or purified SlpA (FIG. 6 and Table 6); however, NCK2187 and SlpAdid not confer any protection in Signr3^(−/−) mice (FIG. 6).Measurements included weight loss, histopathology-based colitis scores,evaluation of gross mucosal damage, and immune cell recruitment andactivation (FIGS. 6 and 7).

Disease progression and inflammation were associated with alterations inintestinal barrier integrity and the composition of the gut microbiota(FIGS. 6E-6H). Several gut permeability markers were evaluated byRT-PCR. The restoration of claudins (Cldnl, Cldn3, and Cldn5) in WT micetreated with NCK2187 or SlpA, indicate that NCK2187 and SlpA werecapable of promoting intercellular tight junctions (FIG. 6E).

Accordingly, only NCK2187 and SlpA treatments of WT mice preventedincreased fecal albumin levels seen with protein-losing enteropathiesafter DSS treatment (FIG. 6F). On the other hand, no positive effects byNCK2187 or SlpA on barrier integrity were noted in Signr3^(−/−) mice(data not shown). In terms of microbiota composition, protected WT mice(NCK2187- and SlpA-treated) clustered together in UniFrac analyses,while diseased untreated and NCK56-treated WT mice clustered separately(FIG. 6G).

Conversely, the microbial communities of all DSS-treated Signr3^(−/−)mice formed a single cluster, indicating that dysbiosis was uniformlydistributed independent of treatment group (FIG. 6G). Furthermore,richness and diversity, analyzed by the Chao Richness index and ShannonDiversity index, respectively, were maintained in NCK2187- andSlpA-treated WT mice, while no such effects could be observed inSignr3^(−/−) mice (FIG. 6H).

TABLE 6 Statistical analysis of weight loss curves in DSS-inducedcolitis in WT mice. Weight Loss (p-value) vs. + DSS alone vs. + NCK56Day +NCK2187 +SlpA +NCK2187 +SlpA 1 1.0 1.0 1.0 1.0 2 0.6705 0.75670.2844 0.7430 3 0.9668 0.7071 0.0862 0.0580 4 0.3974 0.5816 0.83940.2873 5 0.6360 0.2916 0.3444 0.1472 6 0.0087 0.0181 0.0042 0.0098 7<0.0001 0.0001 <0.0001 <0.0001 8 <0.0001 <0.0001 <0.0001 <0.0001 9<0.0001 0.0011 <0.0001 0.0035 10 <0.0001 0.0023 <0.0001 0.0359

Induced immune responses in the colons of DSS-treated mice were analyzedto determine differences, if any, among the treatment groups.Neutrophilic infiltration in the colons of NCK2187- and SlpA-treatedSIGNR3-sufficient mice that were given DSS decreased to nearlyPBS-treated control mice levels; while, in contrast, an even higherfrequency of infiltrating neutrophils was detected in Signr3^(−/−) miceafter the induction of colitis, irrespective of the treatment groupanalyzed (FIG. 7A). Similarly, the number of IL-1β-producing DCs andmacrophages was significantly decreased with NCK2187 and SlpA treatmentin WT mice; however, no changes were observed among the differenttreatment groups in the absence of SlpA:SIGNR3 signaling (FIG. 7B).

Pathogenic inflammation can result in proinflammatory FoxP3⁺RORγt⁺ Tregs(Hovhannisyan et al., 2011; Khazaie et al., 2012). While no major changein the total number of FoxP3⁺ Tregs was measured among the WT andSignr3^(−/−) KO groups, the quality of these Tregs was significantlyaltered. A large number of FoxP3⁺ cells co-expressed RORγt after DSStreatment in both WT and Signr3^(−/−) mice (FIG. 7C). However, inaccordance with the protection observed, NCK2187 and SlpA treatmentprevented the generation of FoxP3+RORγt⁺ Tregs only in WT mice but notSignr3^(−/−) mice (FIG. 7C). Correspondingly, the levels of circulatingcytokines in the sera (IL-1β, IL-10, IL-17A, IFNγ and TNF-α) wererebalanced only in NCK2187- and SlpA-treated WT mice but not inSignr3^(−/−) mice (FIG. 9).

Collectively, these clinical and immunologic data provide robustevidence in support of an immunoregulatory role for L. acidophilus SlpAthat is highly dependent on intact SIGNR3 signaling.

Example 5—Effective Dose for Prevention of DSS-Induced Colitis by L.acidophilus Slpa

To test the required dose of purified SlpA for efficacy in theprevention of colitis, DSS-induced colitis model was employed. 6 oraltreatments with 150 μg SlpA/mouse, and only 4 oral gavages of 10⁹ CFUNCK2187 were sufficient to prevent weight loss in mice given 3% DSS inthe drinking water for 5 days (FIG. 11A). See FIG. 10A for graphicalsummary of the treatment regimen. For the DSS-induced colitis preventionstudies, age and sex-matched C57BL/6 mice were gavaged twice with NCK56(WT), NCK2187, or purified SlpA, prior to being given 3% DSS in thedrinking water. At the start of the treatment, the mice were gavagedtwice more with the different bacterial strains or 4 more times withSlpA to compensate for the ability of NCK56 and NCK2187 to persist inthese animals for 3 days post-gavage (FIG. 1D). Mice treated withNCK2187 or SlpA developed only a mild form of colitis and recoveredsignificantly faster than DSS-alone or NCK56-treated mice (FIG. 11).Collectively, these data suggest that NCK2187 and SlpA are suitabletreatments for the prevention of colitis.

Example 6—Reversal of DSS-Induced Colitis by Acidophilus Slpa

Having tested the protective capacity of L. acidophilus SlpA, itsability to ameliorate established colitis was tested. For these studies,mice were orally gavaged with varying bacteria strains or purified SlpAonce signs of colitis were noted, i.e., diarrhea and fecal occult blood(see FIG. 10B for treatment regimen). Mice receiving either NCK2187 orSlpA recovered significantly faster than NCK56-treated or DSS-alonetreated mice (FIG. 12). These results indicate that NCK2187 and SlpA areefficacious in ameliorating existing colitis.

Example 7—Prevention of T Cell-Induced Colitis by L. acidophilus Slpa

The regulatory effects of L. acidophilus SlpA in a chronic inflammatorymodel of colitis, namely, the pathogenic T cell transfer model wastested. Immunodeficient RagF mice were injected with regulatory T cell(Treg)-depleted CD4⁺ splenic cells (CD4⁺CD25⁻), then left untreated orgavaged with NCK56, NCK2187, or purified SlpA (150 μg/mouse), andmonitored for the onset of colitis. Recipient mice were gavaged twicewith their corresponding treatments at days 1 and 3 after the transfer,then once a week for the next 4 weeks, for a total of 6 gavages. Anadditional group receiving Tregs was used as a positive control for theprevention of colitis. Oral treatment with 10⁹ CFU NCK2187 was aseffective as the Tregs in the prevention of weight loss in recipientRag1^(−/−) mice (FIG. 13A). Purified SlpA was also found to beprotective in recipient Rag1^(−/−) mice (FIG. 13); however, given thechronic nature of the model, a higher dose of SlpA may be needed toreach the level of protection observed with NCK2187.

Example 8—NCK2187 Promotes the Generation of Foxp3⁺ Regulatory T Cells(Tregs)

To gain a better understanding of the protective mechanisms induced byL. acidophilus SlpA that may explain the aforementioned protection, weorally gavaged healthy control mice with either NCK56 or NCK2187, andanalyzed the frequency of Tregs locally and systemically. Compared tountreated or NCK56 treated mice, NCK2187 induced the generation of Tregs(FIG. 14). Taken together, these data suggest that L. acidophilus SlpAtriggers important regulatory signaling cascades that may enable therestoration of intestinal homeostasis in experimental models of colitis.

Example 9

The human GI tract harbors trillions of microbes, most of which arebacteria (Qin et al., 2010), and are critical determinants to the healthof the host (Nicholson et al., 2012; Subramanian et al., 2014). This isespecially true in the case of IBD, given the intimate association ofthe gut microbiota and their gene products with the adjacent colonictissue (Hold et al., 2014; Huttenhower et al., 2014). Early experimentssuggest that susceptibility to pathogenic intestinal inflammation inexperimental colitis depends upon the presence of enteric antigens (Kuhnet al., 1993), and were later supported by human studies, whichdemonstrated that an imbalance in the commensal bacterial composition,termed dysbiosis, is a defining characteristic of patients sufferingfrom IBD (Frank et al., 2007; Sokol et al., 2006). Accordingly, a majorfocus in the field has been the identification of effector bacterialstrains that influence the immune system (Ahern et al., 2014), and thus,may be employed to reprogram undesired immune responses, both locallyand systemically.

Search for microbes with immunoregulatory properties at the strain leveland not merely at the species level is warranted. Certain embodiments ofthe current invention identify specific bacterial molecule-host receptorinteractions that may account for the responses induced by effectorbacterial strains. For example, oral treatment using a L. acidophilusstrain lacking the gene responsible for LTA biosynthesis significantlyreduced pathogenic inflammation in the GI tract, thereby promoting themitigation of induced colitis (Mohamadzadeh et al., 2011) and theablation of colonic polyposis (Khazaie et al., 2012). The bacterialacking LTA and their uses are described in US Patent ApplicationPublication 20130224153, the contents of which are incorporated byreference herein in its entirety. However, a need still remains foridentifying other bacterial strains and/or agents useful for treatmentof inflammatory diseases, such as colitis or inflammatory bowel disease.

To address this need, the current invention provides bacteria withsystematically deleted genes for the construction of novel bacterialstrains, for example, NCK2187, and the assignment of roles to slpcandidate genes that are responsible for SlpA, SlpB, and SlpX proteinexpression (Goh et al., 2009). This molecular approach to targetinggenes in L. acidophilus defined the functional role of SlpA anddemonstrated that SlpA affects intestinal innate cells and conventionalT cell subset activation, including Tregs, in steady-state and murinecolitis models.

As seen in FIGS. 3 and 4, NCK2187 and purified SlpA not only mitigated Tcell-induced colitis by significantly reducing inflammation, but alsoprotected the composition of the microbiota and intestinal barrierfunction. Additionally, systemic immune responses were also altered,whereupon the levels of proinflammatory cytokines, including IL-1β,whose detrimental role in IBD was recently demonstrated (Coccia et al.,2012), decreased significantly. These data suggests the involvement ofthe IL-1β signaling axis in intestinal protection. Accordingly,gene-screening results, along with SlpA binding to SIGNR3, highlight theinvolvement of SIGNR3 in the process of tempering highly activated gutimmune responses. Additional data regarding SIGNR3 engagement usingSignr3^(−/−) mice clarified the role of this signaling molecule ininduced immune regulation, as was also documented in the Leishmaniainfantum murine model (Lefevre et al., 2013).

These data reflect that the SlpA: SIGNR3 interaction significantlyreduces the high affinity receptors for LTB₄ in T cell transferredRag1^(−/−) mice. Downregulation of LTB₄ and/or its receptors is criticalin preventing inflammasome activation, which otherwise results inincreased IL-10 (Lefevre et al., 2013). Interestingly, interrupting theinteraction between SlpA and SIGNR3 resulted in hyperactive immunity andthe production of IL-1β in Signr3^(−/−) mice under inflammatoryconditions. Such dysregulated immune responses in Signr3^(−/−) micepromoted neutrophil infiltration and significantly affected the functionof colonic Tregs, which reverted toward proinflammatory FoxP3⁺ RORγt⁺Tregs, all of which significantly contributed to pathologicinflammation, a condition seen in IBD progression.

In contrast, balanced immunity was restored in WT mice that were treatedwith NCK2187 or SlpA. Induced colonic inflammation in WT mice that weretreated with NCK56, but not in NCK2187- or SlpA-treated mice, and in KOmice, regardless of treatment, resulted in microbial dysbiosis andbarrier dysfunction, another hallmark of IBD.

As such, the current invention indicates that the interaction of SlpAwith SIGNR3 can impact the status of innate and T cell polarization ininduced colitis. Also, effective modulation of these cellular andmolecular factors may significantly modify pathogenic inflammation thatresults in colitis, and would therefore restore intestinal homeostasisby rebalancing deteriorated immunity, the composition of the gutmicrobiota, and mucosal barrier function.

Example 10—A Process for Isolation and Purification of Slpa fromLactobacillus Acidophilus

It is estimated that over 1 million individuals in the U.S. suffer fromIBD (Kappelman et al., 2007; Kappelman et al., 2013). Additionally,ample evidence indicates that dysfunctional immune responses arepotentially elicited by gut dysbiosis (Major et al., 2014). Tospecifically determine the effects of SlpA and its binding to SIGNR3 onintestinal cells and the consequences thereafter, the uppcounter-selective knockout strategy (Mohamadzadeh et al., 2011a) wasused to generate a new strain of L. acidophilus, called NCK2187, whichexpresses only SlpA Our data show that SlpA plays a critical role incontrolling immune responses upon its interaction with SIGNR3, resultingin the diminution of induced colitis, protection of intestinal barrierintegrity, and sustenance of the gut bacterial composition. To buildupon these observations, we have optimized the purification of SlpA toinvestigate its physiological effects when orally administrated to mice,and evaluated whether this protein could resist the harsh condition ofthe gastrointestinal milieu, both important factors that may facilitatethe feasibility of potential clinical trials.

Isolation and Detection of L. acidophilus Surface Layer Protein A

S-layers are paracrystalline (glyco) protein arrays that are present inabundance on the cell surface of a subset of eubacteria and archaea. Wefirst sought to improve the process of SlpA isolation and purification.For this purpose, we used sodium chloride (NaCl) (5 M) as discussed inthe Materials and Methods section. To avoid non-SlpA proteincontamination in our isolation, we employed the LTA-, SlpB-, andSlpX-deficient L. acidophilus NCK2187 strain. Visualization of theisolated protein by SDS-PAGE showed a single protein band of theexpected size (46 kDa, FIG. 15A). An automated mass spectrometrymicrobial identification system that uses Matrix Assisted LaserDesorption Ionization Time-of-Flight technology (MALDI-TOF) indicated 97unique spectra and 55 unique peptides generated post-trypsinization ofthe protein isolate, which identified two possible proteins [gi|58336516(SlpA) and gi|362076610 (SlpB)] (FIG. 15B). Further analyses revealedthat the peptides generated cover 54% of SlpA and 18% of SlpB(highlighted, FIG. 15C). However, the coverage region of SlpB is sharedbetween SlpA and SlpB (red box, FIG. 15C), and no single unique peptidefrom SlpB was identified. MALDI-TOF data were analyzed on Scaffold1(Searle, 2010).26 Therefore, mass spectrometry and SDS-PAGE analysesclearly demonstrated that the identity of the purified SlpA protein wasretained whether purified by NaCl or by LiCl (FIG. 15).

To assess potential toxicity of the isolated SlpA, groups of C57BL/6mice were then orally gavaged with SlpA (0, 150, 300, 600 μg/100 μL permouse) every other day for a total of four times. Subsequently, theblood chemistry profiles of these animals were analyzed. Obtained datademonstrated that oral treatment of the mice with varying doses of SlpAdid not significantly alter whole blood biochemical values in theseanimals (FIG. 16). Changes in enzyme activity or concentration of otheranalytes in the blood were used as metrics of tissue damage orphysiologic stress. Various parameters were measured, including totalprotein and albumin; the concentration of globulins is a calculationbased on the aforementioned measurements (FIG. 16A). Function of theurinary system was evaluated by measuring blood levels of urea nitrogen(BUN) and creatinine, which are normally excreted by the kidneys (FIG.16B). Any evidence of hepatocyte injury was assessed by measuring theactivity of the hepatocellular leakage enzyme, alanine aminotransferase(ALT) (FIG. 16C). The production and activity of alkaline phosphatase(ALP), which is associated with biliary epithelial cells and canalicularmembranes of cells in the liver (Center, 2007),27 can be seen withsolubilization of hepatocyte membranes due to increased bile salts andrelease of membrane blebs with cellular injury (FIG. 16D) (Thrall etal., 2012).28 The electrolytes, sodium, potassium, calcium, andphosphorus, were also measured to gauge any changes in hydration status,excretional activity, or global cellular damage within the treated mice(FIG. 16E). No statistical differences were found in any of theparameters when comparing the controls and those mice receiving varyingdoses of SlpA administration, indicating no evidence of toxicity withoral treatment with SlpA in these animals.

We then elected to generate a specific monoclonal antibody againstpurified SlpA (Bergeron et al., 2009; Simrell et al., 1979).29, 30 Thus,groups of C57BL/6 mice were immunized with purified SlpA with L. gasserias an adjuvant for 3 months (every week/100 μg of SlpA). Subsequently,spleen cells were derived to generate hybridoma cells producingmonoclonal antibody (mAb) recognizing SlpA. As seen in FIG. 17, theantibody derived from one of our hybridoma cell clones, BM1, recognizedSlpA by Western blot (FIG. 17A). Furthermore, this mAb also recognizedSlpA on the surface of SlpA-coated beads, and on SlpA-pulsed RAW 264.7macrophages (FIGS. 17B-C, respectively). As we previously notedsignificant immunomodulatory effects by purified SlpA in the colon,these data suggested that SlpA dissolved in PBS may resist the hostileacidic milieu of the upper gastrointestinal tract and/or enzymaticdegradation within the intestinal lumen. To verify this, we establishedan ELISA using the mAb, BM1, that can detect SlpA (FIG. 17D). Dataclearly show that using this developed ELISA, SlpA can be detected inthe fecal samples from mono-associated germ-free B6 mice (FIG. 17E),indicating that SlpA can potentially resist the harsh conditions of thegastrointestinal system. These data may be useful for initiating Phase Iclinical trials using NaCl-purified SlpA to demonstrate its ability topotentially downregulate induced colonic inflammation in man.

CONCLUDING REMARKS

To gain further insights into the physiological effects of SlpA, studieshave been performed to elucidate the feasibility of Phase I clinicaltrials using this protein. It appears that SlpA using the newly employedpurification method does not elicit potential toxicity when administeredorally to animals, and that the structural epitope(s) of this bacterialprotein can still be recognized by the mAb generated in our laboratoryeven after it is excreted through the feces. Nonetheless, furthermechanistic studies, such as local and peripheral, targeted anduntargeted metabolomics in treated animals, are required to demonstratethe role of SlpA on the host physiology, as well as its effects on otherintestinal immune cells, including epithelial cells, colonic B cells,which mount critical humoral immune responses (e.g., IgA), and innatelymphoid cells (ILCs) in steady state and colonic disease.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

(Genbank Accession No. AAV42337.1) SEQ ID NO: 1   1MERTKSFFKW LTQTKLGFFT IVLVLFWLKT YYIYLTKFNL GAVGPMQQFL LLINPIPSGM  61LLLGIGLFFK GRKSYWIILI IDFLLTLWLF SNILYYREFS NFLSFSIIKT SGSTSDNLGK 121SIAGITLASD FLAFLDIAVI IALLATKVIK MDVRPLKLKV SLLIEFLALS LMGLNLLMAQ 181KDRSGLLTRT FDNNYIVKYL GINEYAIYDG YKTAQTSAQM AKANVSDLKS VRNYLNANKV 241KPNPEYTGVA KGKNVLVIHL ESFQQFLIGY KWKGKEVTPN LNKIYHQKDT ISFDNFFNQV 301GQGKTSDAEM MLENSLYGLQ SGSAMSTYGT SNTFESAPAI LHQQAGYTTA VMHGGAGSFW 361NRNNAYKSFG YQYFMPLSFY ENKPSYYIGY GLKDKIFFDQ SIKYIERLPQ PFYLKMITVT 421NHYPYDIDKK NQSIAKTNTG DETVDGYVQT AHYLDQAIGE LMSWMKKTGL DKKTLIVFYG 481DHYGISGNHH KASAQLLKKK SFNDFDNLQF QRVPLMFHMK GLKGGINHTY GGEIDVLPTL 541LNLLGIKDSD TIQFGYDLLS KNAPQIVAQR NGDFITPEYS KVGSDYYYTK TGKRIKPNKK 601LKAELTAISN TVTTQLSLSD RVINGNLLRF YRPKWFTKVK PKDYDYNKEP SLKRLFNDPS 661KTSLWYQNHK KTTQKDFKTD APELKK (Genbank Accession No. YP_193105)SEQ ID NO: 2    1MKKNLRIVSA AAAALLAVAP VAASAVSTVN AAAVNAIAVG GSATPLPNNS DVQISSSVAG  61VTTKNGSSYT NGRISGSINA SYNGTSYSAN FSSSNAGVVV STPGHTELSG EQINGLEPGS 121AVTVTLRDGV SFNFGSTNAN KTITLAFPKN VSAAGLADAN KVSATSETSV DAGKTIQVKT 181DKNGVVSFGS AQVLNVKVVE TSDVRAVSFY DIQTGKTVEN GTLSIVAGSN ARANVQEIVN 241AFNAKYQASQ LNNANSNANV RLTDNNAQAV ATMLRAQNID VDAQGYFTAP ASLSLTFHAE 301STQNNETAQL PVTVSVTNGK EVTPSTVDSV SKRIMHNAYY YDKDAKRVGT DSVKRYNSVS 361VLPNTTTING KAYYQVVENG KAVDKYINAA NIDGTKRTLK HNAYVYASSK KRANKVVLKK 421GEVVTTYGAS YTFKNGQKYY KIGDNTDKTY VKVANFR(Genbank Accession No. YP_193425) SEQ ID NO: 3   1MKKNRKMLGL AAATLLAVAP VATSVVPVQA DTAVNVGSAA GTGANTTNTT TQAPQNKPYF  61TYNNEIIGEA TQSNPLGNVV RTTISFKSDD KVSDLISTIS KAVQFHKNNS ASGENVTINE 121NDFINQLKAN GVTVKTVQPS NKNEKAYEAI DKVPSTSFNI TLSATGDNNQ TATIQIPMVP 181QGASTPTDTT QNPQINWTKG GQAQSSSLNG QVFQVAVGSN FNPLNFTNSN GENIIVSAQQ 241SKNNTTFASI EATSNPVNTS EAGRYYNVTL TATGNTGKKT TATYTVLITS SQKQTLYGNG 301ESTISTYSIY GNNVLSNSTT FKDGDQVYVS DQTKTVGGVS YSQVSPKSKN DANSSNIWVK 361TSALVKPAGD TNVKTYPVMV DSRAYDKNGN YLGHMYYAYD NIDIVPTVVT INGKTYYKVA 421NKDEYVRVTN ITGNQRTLKH NAYIYWSSYR RTPGTGKMYR GQTVTTYGPQ MKFKNGKKYY 481RIEGCRNNNK RYIKAVNFY (Genbank Accession No. P35829) SEQ ID NO: 4   1MKKNLRIVSA AAAALLAVAP VAASAVSTVS AATTINASSS AINTNTNAKY DVDVTPSVSA  61VAANTANNTP AIAGNLTGTI SASYNGKTYT ANLKADTENA TITAAGSTTA VKPAELAAGV 121AYTVTVNDVS FNFGSENAGK TVTLGSANSN VKFTGTNSDN QTETNVSTLK VKLDQNGVAS 181LTNVSIANVY AINTTDNSNV NFYDVTSGAT VTNGAVSVNA DNQGQVNVAN VVAAINSKYF 241AAQYADKKLN TRTANTEDAI KAALKDQKID VNSVGYFKAP HTFTVNVKAT SNTNGKSATL 301PVVVTVPNVA EPTVASVSKR IMHNAYYYDK DAKRVGTDSV KRYNSVSVLP NTTTINGKTY 361YQVVENGKAV DKYINAANID GTKRTLKHNA YVYASSKKRA NKVVLKKGEV VTTYGASYTF 421KNGQKYYKIG DNTDKTYVKV ANFR Uniprot Access Number C2HR60 SEQ ID NO: 47AATTINASSSAINTNTNAKYDVDVTPSVSAVAAVAANTANNTPAIAGNLTGTISASYNGKTYTANLKADTENATITAAGSTTAVKPAELAAGVAYTVTVNDVSFNFGSENAGKTVTLGSANSNVKFTGTNSDNQTETNVSTLKVKLDQNGVASLTNVSIANVYAINTTDNSNVNFYDVTSGATVTNGAVSVNADNQGQVNVANVVAAINSKYFAAQYADKKLNTRTANTEDAIKAALKDQKIDVNSVGYFKAPHTFTVNVKATSNTNGKSATLPVVVTVPNVAEPTVAUniprot Access Number P35829 SEQ ID NO: 48MKKNLRIVSAAAAALLAVAPVAASAVSTVSAATTINASSSAINTNTNAKYDVDVTPSVSAVAANTANNTPAIAGNLTGTISASYNGKTYTANLKADTENATITAAGSTTAVKPAELAAGVAYTVTVNDVSFNFGSENAGKTVTLGSANSNVKFTGTNSDNQTETNVSTLKVKLDQNGVASLTNVSIANVYAINTTDNSNVNFYDVTSGATVTNGAVSVNADNQGQVNVANVVAAINSKYFAAQYADKKLNTRTANTEDAIKAALKDQKIDVNSVGYFKAPHTFTVNVKATSNTNGKSATLPVVVTVPNVAEPTVASVSKRIMHNAYYYDKDAKRVGTDSVKRYNSVSVLPNTTTINGKTYYQVVENGKAVDKYINAANIDGTKRTLKHNAYVYASSKKRANKVVLKKGEVVTTYGASYTFKNGQKYYKIGDNTDKTYVKVANFR Uniprot Access Number G1UE81 SEQ ID NO: 49MFGGRKIMQSSLKKSLYLGLAALSFAGVAAVSTTASAKSYATAGAYTTLKTDATKRNVEATGTNALYTKPGTVKGAKVVASKATMAKLASSKKSADYFRAYGVKTTNRGSVYYRVVSMDGKYRGYVYGGKSDTAFAGGIKSADTTTTATTPTRTTGYYLKDVSKNTLWTAPKNTQYKASKVSLYGVKSTDTFKVDSAATKTREGSLYYHVTDTQNTSVSGWIYAGKGYVAGATTQDLGGLSLTMSDAAATSDNSVKVVYRASGSQVGTATWVTAAAGTKAGATVGTTAVNAAGVKLADFVTNSLPSGYTTTGTVDTASATYGNTVYVDVTAAATSKVQLVADNVDNTASTTDNAVAGVLANGAKLSSSDLSATLKEAGIKALTGTKGEAIGATNLATISGAFDTAEINGSKTYYAANGDAYHYVFTYEPANFANDNRLATYGDTLTASFKAVLTKGAPSASSSNSSWIAUniprot Access Number Q9Z4J9 SEQ ID NO: 50MKKNLRIVSAAAAALLAVAPVAASAVSVNAADNTVATTTNTANTVINADGTAINTPADAKYDVDVTPNLTATAASTVNGQTINGSITGNITASYNGQSYTGTLDTKNGKVSVADSKGTAVTDFSKLTNGSYTVTVSGVSFNFGTANANKTITLGSKNSNVKFAGADGKFADTVKVELGQNGTLTTPISVQVSNVNALDLSNANGVNFYNASNGSQVTKGSVNVTAGLIGRLNVSTVASEILKNCAAYQVSNGKPVSQLPDQKAVVADVNAALKAANIPVDNAGWFTAPISLSVNVKASSSINGVGCYFTCTVNVANGKDMTVPSQSKTIMHNAYYYDKDAKRVGTDKLTRYNSVTVAMNTTTINGKAYYEVIENGKATGKFINADNIDGTKRTLKHNAYVYKTSKKRANKVTLKKGTEVTTYGGTYTFKNGKQYYKIGNNTDKTYVKASNF Uniprot Access Number H6VTN4SEQ ID NO: 51MKKNLRIVSAAAAALLAVAPVAAAGVSSVTASSIEFVGSSNSSLLPEVNDHTVNFGINFNAIGAYGNVPSSVSATAEVTINGQKTTINLPENQKSYIYYATTNESVDASKLVAGQKYYTGINNASLNLGSPNHDKDITLEGSNVSFKTNDSDPYTKTLKVNTDKNGVISNLSIKSANFDAVDVNNARTVSFYDADTGNIVTSGALEINAGPNAQMNVQTILAKFEQKYQAAQLNNAGTTNNVSYNNDLISTTPADLAAQLKKAGYSVDNNGYFTAKHSFTVNFSAKSGQNGYTTTMPVTVTVPNVAEETVPSQIRTVMHNAFFYDKNGKRVGSDKVTRYNSATVAMNTTTIIGKAYYEVIENGKATGKFINAANIDGTKRTLKHNAYVYKSSKKRANKVVLKKGETVVTYGGAYTFKNGKQYYKIGNNTDKTYVKVANF Uniprot Access Number Q09FM2 SEQ ID NO: 52MKKNLRIVSAAAAALLAVAPVAASAVSVNAASSSAVQTATNIGTVLPLTDGSTVNVKPNISLNTSAYEGVKANISVSFSATVDGTTATSNFTPNASTIELWKNEKNKVTQVTYLQQVTSSNAGATYQVKMTQVGLNFGSQNANKKVTLTFPEGDMFKTADTSLAQSHEVKLDQNGTITLPEVVMNVTAKDFANPAVVNWYNTATNAVVSTGNIELFAGSDAGKMNVAQVVSATEKKYHASNYGTKANQESSTISYTNNLKDALKAMNVDVDAQGWFVAPKSFTFNMTAKANNNDASSTLAVTVSVPNGKDMTVPSQSKTVMHNAFFYDKNGKRVGSDKVTRYNSATVAMNTTTINGKAYYEVIENGKATGKFINAANIDGTKRTLKHNAYVYKSSKKRANKVVLKKGTEVVTYGGAYTFKNGKQYYKIGNNTDKTYVKVSNF Uniprot Access Number L7YE91 SEQ ID NO: 53SVSESKDTVNVTPSFTLTSAIPAKGVPATLQGSIEASLNGTSVTADVADVAKDVTLTDGNKTVYSYNERENKVDNNLSAVEASKEYTMTLSGVGFSFGKANAGKTLTFKLPKNVKVNDTSNDVKVSLDQYGNATNLKFVISNIKAYDSANTNAVSFYAAKSGLVATQGSYMTLADENGNLNVNTLLDKLKGKYEAMQFKDSKFETVNVNTTADDVKAELEKAGIKVDAANNFEAPDTFTVTLNAKSDVNGKTASLPVVVTVPNGKSTVVPSQSKTIMHNAYYYDKDAKRVGTDKVTRYNAVTVAMNTTKLANGISYYEVIEN Uniprot Access Number K8DVK7 SEQ ID NO: 54ADSAINANTNAKYDVDVTPSISAIAAVAKSDTMPAIPGSLTGSISASYNGKSYTANLPKDSGNATITDSNNNTVKPAKLEADKAYTVTVPDVSFNFGSENAGKVITIGSANPNVTFTKKTGDQPASTVKVTLDQDGVAKLSSVQIKNVYAIDTTYNSNVNFYDVTTGAIVTTGAVSIDADNQGQLNITSVVAAINSKYFAAQYDKKQLTNDVTFDTETAVKDALKAQKIEVSSVGYFKAPHTFTVNVKATSNKNGKSATLPVTVTVPNVADPVVPSQSKTIMHNAYFYDKDAKRVGTDKVTRYNTVTVAMNTTKLANGISYYEVIENGKA Uniprot Access Number F0NUB7SEQ ID NO: 55MDHVSKGFVHYRLLSHAEPMAYYIFYISRRKDHMKKNLRIVSAAAAALLAVAPVAATAMPVNAATTINADSAINANTNAKYDVDVTPSISAIAKVTGSATIPGSLTGSISASYNGKSYTANLPKDSGNATIADKHGNPVKPADLEADKAYTVTVPDVSFNFGSENAGKEITIGSANQNVTFTTKDSQSGSTVSGSTVKVTLDQDGVAKLSSVQIKDVYAIDTTYNSNVNFYDVTTGAIVTTGAVSIDADNQGQLNTASVVAAISSKYFAAQYADKNLTSDNVTYNIETAVKDALKAQKIEVSSVGYFKAPHTFTVNVKATSNKNGKSATLPVTVTVPNVADPVVPSQSKTIMHNAYFYDKDAKRVGTDKVTRYNTVTVAMNTTKLANGISYYEVIENGKATGKYINADNIDGTKRTLKHNAYVYKTSKKRANKVVLKKGTEVTTYGGSYKFKNGKKYYKIGADTKKTYVRVENFD

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We claim:
 1. A recombinant lactic acid bacterium that is geneticallymodified to express a polypeptide comprising SEQ ID NO: 4 or apolypeptide that is at least 95% identical to SEQ ID NO: 4 and does notexpress (i) a polypeptide comprising SEQ ID NO: 1 or orthologs thereofor a polypeptide having at least 95% sequence identity to SEQ ID NO: 1;(ii) a polypeptide comprising SEQ ID NO: 2 or orthologs thereof or apolypeptide having at least 95% sequence identity to SEQ ID NO: 2; and(iii) a polypeptide comprising SEQ ID NO: 3 or orthologs thereof or apolypeptide having at least 95% sequence identity to SEQ ID NO:
 3. 2.The recombinant lactic acid bacterium according to claim 1, wherein saidrecombinant bacterium is a Lactobacillus strain.
 3. The recombinantlactic acid bacterium according to claim 2, wherein the Lactobacillusstrain is L. lactis.
 4. The recombinant lactic acid bacterium accordingto claim 1, wherein said bacterium lacks genes encoding SEQ ID NO: 1 ororthologues thereof, SEQ ID NO: 2 or orthologues thereof, and SEQ ID NO:3 or orthologues thereof.
 5. The recombinant lactic acid bacteriumaccording to claim 1, wherein said bacterium does not express (i) apolypeptide having at least 95% sequence identity to SEQ ID NO: 1; (ii)a polypeptide having at least 95% sequence identity to SEQ ID NO: 2; and(iii) a polypeptide having at least 95% sequence identity to SEQ ID NO:3.
 6. A probiotic food comprising the recombinant lactic acid bacteriumof claim 1.